SMART MODE:

NORMAL GENOMIC

• Kranewitter WJ, Ylanne J, Gimona M
• UNC-87 is an actin-bundling protein.
• J Biol Chem. 2001; 276: 6306-12
• Display abstract

• The Caenorhabditis elegans unc-87 gene product is essential for the maintenance of the nematode body wall muscle where it is found colocalized with actin in the I band. The molecular domain structure of the protein reveals similarity to the C-terminal repeat region of the smooth muscle actin-binding protein calponin. In this study we investigated the in vitro function of UNC-87 using both the full-length recombinant molecule and several truncated mutants. According to analytical ultracentrifugation UNC-87 occurs as a monomer in solution. UNC-87 cosedimented with both smooth and skeletal muscle F-actin, but not with monomeric G-actin, and exhibited potent actin filament bundling activity. Actin binding was independent of the presence of tropomyosin and the actin cross-linking proteins filamin and alpha-actinin. Consistent with its actin bundling activity in vitro, UNC-87 tagged with green fluorescent protein associated with and promoted the formation of actin stress fiber bundles in living cells. These data identify UNC-87 as an actin-bundling protein and highlight the calponin-like repeats as a novel actin-binding module.

• Fontao L, Geerts D, Kuikman I, Koster J, Kramer D, Sonnenberg A
• The interaction of plectin with actin: evidence for cross-linking of actin filaments by dimerization of the actin-binding domain of plectin.
• J Cell Sci. 2001; 114: 2065-76
• Display abstract

• Plectin is a major component of the cytoskeleton and is expressed in a wide variety of cell types. It plays an important role in the integrity of the cytoskeleton by cross-linking the three filamentous networks and stabilizing cell-matrix and cell-cell contacts. Sequence analysis showed that plectin contains a highly conserved actin-binding domain, consisting of a pair of calponin-like subdomains. Using yeast two-hybrid assays in combination with in vitro binding experiments, we demonstrate that the actin-binding domain of plectin is fully functional and preferentially binds to polymeric actin. The sequences required for actin binding were identified at the C-terminal end of the first calponin homology domain within the actin-binding domain of plectin. We found that the actin-binding domain of plectin is able to bundle actin filaments and we present evidence that this is mediated by the dimerization of this domain. In addition we also show that plectin and another member of the plakin family, dystonin, can heterodimerize by their actin-binding domains. We propose a new mechanism by which plectin and possibly also other actin-binding proteins can regulate the organization of the F-actin network in the cell.

• Howes EA, Hurst SM, Jones R
• Actin and actin-binding proteins in bovine spermatozoa: potential role in membrane remodeling and intracellular signaling during epididymal maturation and the acrosome reaction.
• J Androl. 2001; 22: 62-72
• Display abstract

• The actin cytoskeleton influences a wide range of functions in nonmuscle somatic cells, including shape, movement, and interactions with extracellular matrices. The role of actin in mammalian male germ cells, however, particularly during post-testicular development, is not well understood. In this paper, we examine 1) the distribution of 3 actin-regulatory proteins (thymosin beta10, destrin, and a testis-specific actin capping protein) involved in controlling the balance between actin monomers (G-actin) and actin filaments (F-actin), and 2) the distribution and polymerization status of actin in bull spermatozoa during epididymal maturation and following acrosomal exocytosis. Results show that in fixed, permeabilized testicular spermatozoa all 3 regulatory proteins (as determined by binding of specific antibodies) are localized primarily to the acrosomal domain but during epididymal maturation they become confined to the equatorial segment. Following ejaculation, however, they extend back into the acrosomal region. In spermatozoa induced to undergo an acrosome reaction with the calcium ionophore, A23187, further rearrangement occurs with destrin, thymosin beta10, and TS-ACP appearing in the postacrosomal domain. Actin is also found over the acrosome of testicular spermatozoa with both G- and F-actin present, although the 2 forms show slightly different patterns of distribution. Subsequently, actin in the sperm head is largely confined to the equatorial segment until F-actin appears in the postacrosomal domain of acrosome-reacted spermatozoa. This redistribution of actin and actin-regulatory proteins, coupled with changing levels of actin polymerization, suggest a continuing role for actin in both post-testicular sperm maturation and acrosomal exocytosis.

• van der Flier A, Sonnenberg A
• Structural and functional aspects of filamins.
• Biochim Biophys Acta. 2001; 1538: 99-117
• Display abstract

• Filamins are a family of high molecular mass cytoskeletal proteins that organize filamentous actin in networks and stress fibers. Over the past few years it has become clear that filamins anchor various transmembrane proteins to the actin cytoskeleton and provide a scaffold for a wide range of cytoplasmic signaling proteins. The recent cloning of three human filamins and studies on filamin orthologues from chicken and Drosophila revealed unexpected complexity of the filamin family, the biological implications of which have just started to be addressed. Expression of dysfunctional filamin-A leads to the genetic disorder of ventricular heterotopia and gives reason to expect that abnormalities in the other isogenes may also be connected with human disease. In this review aspects of filamin structure, its splice variants, binding partners and biological function will be discussed.

• Volkmann N, DeRosier D, Matsudaira P, Hanein D
• An atomic model of actin filaments cross-linked by fimbrin and its implications for bundle assembly and function.
• J Cell Biol. 2001; 153: 947-56
• Display abstract

• Actin bundles have profound effects on cellular shape, division, adhesion, motility, and signaling. Fimbrin belongs to a large family of actin-bundling proteins and is involved in the formation of tightly ordered cross-linked bundles in the brush border microvilli and in the stereocilia of inner ear hair cells. Polymorphism in these three-dimensional (3D) bundles has prevented the detailed structural characterization required for in-depth understanding of their morphogenesis and function. Here, we describe the structural characterization of two-dimensional arrays of actin cross-linked with human T-fimbrin. Structural information obtained by electron microscopy, x-ray crystallography, and homology modeling allowed us to build the first molecular model for the complete actin-fimbrin cross-link. The restriction of the arrays to two dimensions allowed us to deduce the spatial relationship between the components, the mode of fimbrin cross-linking, and the flexibility within the cross-link. The atomic model of the fimbrin cross-link, the cross-linking rules deduced from the arrays, and the hexagonal packing of actin bundles in situ were all combined to generate an atomic model for 3D actin-fimbrin bundles. Furthermore, the assembly of the actin-fimbrin arrays suggests coupling between actin polymerization, fimbrin binding, and crossbridge formation, presumably achieved by a feedback between conformational changes and changes in affinity.

• McGough A, Pope B, Weeds A
• The ADF/cofilin family: accelerators of actin reorganization.
• Results Probl Cell Differ. 2001; 32: 135-54

• Ono S et al.
• The C-terminal tail of UNC-60B (actin depolymerizing factor/cofilin) is critical for maintaining its stable association with F-actin and is implicated in the second actin-binding site.
• J Biol Chem. 2001; 276: 5952-8
• Display abstract

• Actin depolymerizing factor (ADF)/cofilin changes the twist of actin filaments by binding two longitudinally associated actin subunits. In the absence of an atomic model of the ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin that are essential for filament binding. Here, we have characterized the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a novel determinant for its association with F-actin. Removal of the C-terminal isoleucine (Ile152) by carboxypeptidase A or truncation by mutagenesis eliminated F-actin binding activity but strongly enhanced actin depolymerizing activity. Replacement of Ile152 by Ala had a similar but less marked effect; F-actin binding was weakened and depolymerizing activity slightly enhanced. Truncation of both Arg151 and Ile152 or replacement of Arg151 with Ala also abolished F-actin binding and enhanced depolymerizing activity. Loss of F-actin binding in these mutants was accompanied by loss or greatly decreased severing activity. All of the variants of UNC-60B interacted with G-actin in an indistinguishable manner from wild type. Cryoelectron microscopy showed that UNC-60B changed the twist of F-actin to a similar extent to vertebrate ADF/cofilins. Helical reconstruction and structural modeling of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might be involved in one of the two actin-binding sites.

• Gerber SH, Garcia J, Rizo J, Sudhof TC
• An unusual C(2)-domain in the active-zone protein piccolo: implications for Ca(2+) regulation of neurotransmitter release.
• EMBO J. 2001; 20: 1605-19
• Display abstract

• Ca(2+) regulation of neurotransmitter release is thought to require multiple Ca(2+) sensors with distinct affinities. However, no low-affinity Ca(2+) sensor has been identified at the synapse. We now show that piccolo/aczonin, a recently described active-zone protein with C-terminal C(2)A- and C(2)B-domains, constitutes a presynaptic low-affinity Ca(2+) sensor. Ca(2+) binds to piccolo by virtue of its C(2)A-domain via an unusual mechanism that involves a large conformational change. The distinct Ca(2+)-binding properties of the piccolo C(2)A- domain are mediated by an evolutionarily conserved sequence at the bottom of the C(2)A-domain, which may fold back towards the Ca(2+)-binding sites on the top. Point mutations in this bottom sequence inactivate it, transforming low-affinity Ca(2+) binding (100-200 microM in the presence of phospholipids) into high-affinity Ca(2+) binding (12-14 microM). The unusual Ca(2+)-binding mode of the piccolo C(2)A-domain reveals that C(2)-domains are mechanistically more versatile than previously envisaged. The low Ca(2+) affinity of the piccolo C(2)A-domain suggests that piccolo could function in short-term synaptic plasticity when Ca(2+) concentrations accumulate during repetitive stimulation.

• Moroz OV et al.
• The three-dimensional structure of human S100A12.
• Acta Crystallogr D Biol Crystallogr. 2001; 57: 20-9
• Display abstract

• The crystal structure of human EF-hand calcium-binding protein S100A12 in its calcium-bound form has been determined to 1.95 A resolution by molecular replacement using the structure of the S100B protein. The S100 family members are homologous to calmodulin and other related EF-hand calcium-binding proteins. Like the majority of S100 proteins, S100A12 is a dimer, with the interface between the two subunits being composed mostly of hydrophobic residues. The fold of S100A12 is similar to the other known crystal and solution structures of S100 proteins, except for the linker region between the two EF-hand motifs. Sequence and structure comparison between members of the S100 family suggests that the target-binding region in S100A12 is formed by the linker region and C-terminal residues of one subunit and the N-terminal residues of another subunit of the dimer. The N-terminal region of the target-binding site includes two glutamates that are conserved in most of the S100 sequences. The comparison also provided a better understanding of the role of the residues important for intra- and inter-subunit hydrophobic interactions. The precise role of S100A12 in cell behaviour is yet undefined, as is the case for the whole family, although it has been shown that the interaction of S100A12 with the RAGE receptor is implicated in inflammatory response.

• Bourne Y, Dannenberg J, Pollmann V, Marchot P, Pongs O
• Immunocytochemical localization and crystal structure of human frequenin (neuronal calcium sensor 1).
• J Biol Chem. 2001; 276: 11949-55
• Display abstract

• Frequenin, a member of a large family of myristoyl-switch calcium-binding proteins, functions as a calcium-ion sensor to modulate synaptic activity and secretion. We show that human frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4Kbeta, the mammalian homolog of the yeast kinase PIK1. In addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 A resolution. The overall fold of frequenin resembles those of neurocalcin and the photoreceptor, recoverin, of the same family, with two pairs of calcium-binding EF hands and three bound calcium ions. Despite the similarities, however, frequenin displays significant structural differences. A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin. This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand.

• Tu Y, Huang Y, Zhang Y, Hua Y, Wu C
• A new focal adhesion protein that interacts with integrin-linked kinase and regulates cell adhesion and spreading.
• J Cell Biol. 2001; 153: 585-98
• Display abstract

• Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH(2)-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti-CH-ILKBP antibodies, and green fluorescent protein-CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP-ILK-PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.

• McGhie EJ, Hayward RD, Koronakis V
• Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin.
• EMBO J. 2001; 20: 2131-9
• Display abstract

• Pathogen-induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmonella by non-phagocytic intestinal epithelial cells. Two Salmonella actin-binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F-actin bundling, we demonstrate that SipA stimulates substantially SipC-mediated nucleation of actin polymerization. SipA additionally enhances SipC-mediated F-actin bundling, and SipC-SipA collaboration generates stable networks of F-actin bundles. The data show that bacterial SipC and SipA cooperate to direct efficient modulation of actin dynamics, independently of host cell proteins. The ability of SipA to enhance SipC-induced reorganization of the actin cytoskeleton in vivo was confirmed using semi-permeabilized cultured mammalian cells.

• Zhang Q, Li Y, Howard TH
• Human lymphocyte-specific protein 1, the protein overexpressed in neutrophil actin dysfunction with 47-kDa and 89-kDa protein abnormalities (NAD 47/89), has multiple F-actin binding domains.
• J Immunol. 2000; 165: 2052-8
• Display abstract

• Human lymphocyte-specific protein 1 (LSP1) is an F-actin binding protein, which has an acidic N-terminal half and a basic C-terminal half. In the basic C-terminal half, there are amino acid sequences highly homologous to the actin-binding domains of two known F-actin binding proteins: caldesmon and the villin headpieces (CI, CII, VI, VII). However, the exact numbers and locations of the F-actin binding domains within LSP1 are not clearly defined. In this report, we utilized 125I-labeled F-actin ligand blotting and high-speed F-actin cosedimentation assays to analyze the F-actin binding properties of truncated LSP1 peptides and to define the F-actin binding domains. Results show that LSP1 has at least three and potentially a fourth F-actin binding domain. All F-actin binding domains are located in the basic C-terminal half and correspond to the caldesmon and villin headpiece homologous regions. LSP1 181-245 and LSP1 246-295, containing sequences homologous to caldesmon F-actin binding site I and II, respectively (CI, CII), binds F-actin; similarly, LSP1 306-339 can bind F-actin and contains two inseparable villin headpiece-like F-actin binding domains (VI, VII). Although LSP1 1-305, which does not contain VI and VII regions, retains F-actin binding activity, its binding affinity for F-actin is much weaker than that of full-length LSP1. Site-directed mutagenesis of the basic amino acids in the KRYK (VI) or KYEK (VII) sequences to acidic amino acids create mutants that bind F-actin with lower affinity than full-length wild-type LSP1. High KCl concentrations decrease full-length LSP1 binding to F-actin, suggesting the affinity between LSP1 and F-actin is mainly through electrostatic interaction.

• Peng YF et al.
• Ankycorbin: a novel actin cytoskeleton-associated protein.
• Genes Cells. 2000; 5: 1001-8
• Display abstract

• BACKGROUND: Actin cytoskeleton structures are essential for a wide variety of cell functions, including cell shape change, cell motility, cell adhesion, cell polarity and cytokinesis. Many actin filament (F-actin)-binding proteins have been isolated and implicated in the maintenance and reorganization of actin cytoskeleton structures. RESULTS: We purified here a novel protein with a molecular mass of about 125 kDa (p125) from rat liver. We cloned its cDNA from a mouse kidney cDNA library and determined its nucleotide and deduced amino acid sequences. p125 was a protein of 979 amino acids with a calculated Mr of 108 847. p125 contained six ankyrin repeats in the N-terminal region and a domain predicted to form a coiled-coil structure in the C-terminal region. We named p125 ankycorbin (ankyrin repeat- and coiled-coil structure-containing protein). Northern blot analysis indicated that ankycorbin was ubiquitously expressed in all the tissues examined. Immunofluorescence and immunoelectron microscope analyses revealed that ankycorbin was associated with the cortical actin cytoskeleton structures in terminal web and cell-cell adhesion sites and stress fibres. However, ankycorbin did not directly bind to F-actin as estimated by the F-actin co-sedimentation assay. CONCLUSIONS: These results indicate that ankycorbin is indirectly associated with the actin cytoskeleton structures, presumably through an unidentified factor and suggest that it is involved in their maintenance and/or reorganization.

• Weed SA et al.
• Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex.
• J Cell Biol. 2000; 151: 29-40
• Display abstract

• Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain-containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.

• Pearson MA, Reczek D, Bretscher A, Karplus PA
• Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain.
• Cell. 2000; 101: 259-70
• Display abstract

• The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin.

• Puius YA, Fedorov EV, Eichinger L, Schleicher M, Almo SC
• Mapping the functional surface of domain 2 in the gelsolin superfamily.
• Biochemistry. 2000; 39: 5322-31
• Display abstract

• The crystal structure of the F-actin binding domain 2 of severin, the gelsolin homologue from Dictyostelium discoideum, has been determined by multiple isomorphous replacement and refined to 1.75 A resolution. The structure reveals an alpha-helix-beta-sheet sandwich similar to the domains of gelsolin and villin, and contains two cation-binding sites, as observed in other domain 1 and domain 2 homologues. Comparison of the structures of several gelsolin family domains has identified residues that may mediate F-actin binding in gelsolin domain 2 homologues. To assess the involvement of these residues in F-actin binding, three mutants of human gelsolin domain 2 were assayed for F-actin binding activity and thermodynamic stability. Two of the mutants, RRV168AAA and RLK210AAA, demonstrated a lowered affinity for F-actin, indicating a role for those residues in filament binding. Using both structural and biochemical data, we have constructed a model of the gelsolin domain 1-domain 2-F-actin complex. This model highlights a number of interactions that may serve as positive and negative determinants of filament end- and side-binding.

• Watanabe A, Yonemura I, Gonda K, Numata O
• Cloning and sequencing of the gene for a Tetrahymena fimbrin-like protein.
• J Biochem (Tokyo). 2000; 127: 85-94
• Display abstract

• Tetrahymena F-actin-binding protein, which induces bundling of Tetrahymena F-actin, was localized to a division furrow during cytokinesis. We report here the cloning and characterization of the gene and cDNA of a Tetrahymena F-actin-binding protein. The cDNA encodes a protein comprising 579 deduced amino acids with a calculated molecular mass of 65.9 kDa. The predicted amino acid sequence shares 37.7, 41.8, and 39% identity with the sequences of yeast fimbrin, Arabidopsis thaliana fimbrin, and Dictyostelium discoideum plastin, respectively. The Tetrahymena F-actin-binding protein also shares two actin-binding domains previously identified in the fimbrin/plastin family, but lacks the EF-hand Ca2+-binding motif, suggesting that this protein is a novel-fimbrin-like protein in Tetrahymena. Moreover, we cloned a genomic DNA encoding the Tetrahymena fimbrin-like protein and performed Southern and Northern hybridizations. The results indicate that the genomic DNA possesses 9 introns and that both the gene and transcript of Tetrahymena fimbrin-like protein are single. Thus, we suggest that Tetrahymena fimbrin-like protein localizes to the division furrow and probably cross-links actin filaments in a Ca(2+)-insensitive manner during cytokinesis.

• Llorca O et al.
• Eukaryotic type II chaperonin CCT interacts with actin through specific subunits.
• Nature. 1999; 402: 693-6
• Display abstract

• Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP-1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and alpha-actin by cryo-electron microscopy and image processing. This shows that alpha-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT-substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTdelta and the large domain to CCTbeta or CCTepsilon (both in position 1,4 with respect to delta). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.

• Lim RW, Furukawa R, Fechheimer M
• Evidence of intramolecular regulation of the Dictyostelium discoideum 34 000 Da F-actin-bundling protein.
• Biochemistry. 1999; 38: 16323-32
• Display abstract

• Intramolecular interaction within the Ca(2+)-regulated 34 kDa actin-bundling protein from Dictyostelium discoideum was found to contribute to the regulation of its actin-binding activity. Recombinant N-terminally truncated proteins aa77-295, 124-295, and 139-295 bound actin at > or = 2:1 stoichiometry, which is 5-fold greater than the intact protein aa1-295 as assessed by cosedimentation with F-actin. These proteins also have enhanced cross-linking activity as assessed by viscometry and electron microscopy. All truncated 34 kDa proteins failed to bind (45)Ca(2+) on blots and displayed Ca(2+)-insensitive binding with actin, although most proteins possessed intact putative EF-hand Ca(2+)-binding motifs. An intramolecular interaction within the 34 kDa protein was inferred from direct demonstrations of domain-domain interaction among the truncated 34 kDa proteins both in the presence and absence of actin. The intramolecular interaction between interaction zone 1 (aa71-123) and interaction zone 2 (aa193-254) is proposed to maintain the N-terminal inhibitory region (aa1-76) in close proximity with the strong actin-binding site (aa193-254) in order to modulate the interaction of the intact protein with actin filaments.

• Kusano K, Abe H, Obinata T
• Detection of a sequence involved in actin-binding and phosphoinositide-binding in the N-terminal side of cofilin.
• Mol Cell Biochem. 1999; 190: 133-41
• Display abstract

• Cofilin is an actin-binding protein of low molecular weight which is widely distributed in eukaryotes and is deeply involved in the dynamics of actin assembly in the cytoplasm. The actin-binding ability of cofilin is inhibited by inositol phosphates (PIP2), and the PIP2- and actin-binding site(s) has been localized in residues W104-M115 of the cofilin primary sequence (Yonezawa et al. 1991 ). In the present study, in order to further clarify the functional domains in cofilin molecule, we constructed expression vectors containing cDNAs of different size with deletion at the 3'-region of the open reading frame. The truncated cofilin molecules produced in E. coli were purified and examined for their actin-binding and PIP2-binding ability. We found that the truncated cofilin molecule without C-terminal residues #100-#166 including the previously-described actin-binding site could be cross-linked with actin by EDC, a zero-length cross-linker. In addition, these truncated peptides as well as synthetic peptides corresponding to the N-terminal sequence of cofilin suppressed the inhibitory action of PIP2 on actin-cofilin interaction. These results strongly suggest that additional actin- and PIP2-binding sites exist in the N-terminal region of cofilin.

• Keep NH, Norwood FL, Moores CA, Winder SJ, Kendrick-Jones J
• The 2.0 A structure of the second calponin homology domain from the actin-binding region of the dystrophin homologue utrophin.
• J Mol Biol. 1999; 285: 1257-64
• Display abstract

• Utrophin is a close homologue of dystrophin, the protein defective in Duchenne muscular dystrophy. Like dystrophin, it is composed of three regions: an N-terminal region that binds actin filaments, a large central region with triple coiled-coil repeats, and a C-terminal region that interacts with components in the dystroglycan protein complex at the plasma membrane. The N-terminal actin-binding region consists of two calponin homology domains and is related to the actin-binding domains of a superfamily of proteins including alpha-actinin, spectrin and fimbrin. Here, we present the 2.0 A structure of the second calponin homology domain of utrophin solved by X-ray crystallography, and compare it to the other calponin homology domains previously determined from spectrin and fimbrin.

• Vardar D, Buckley DA, Frank BS, McKnight CJ
• NMR structure of an F-actin-binding "headpiece" motif from villin.
• J Mol Biol. 1999; 294: 1299-310
• Display abstract

• A growing family of F-actin-bundling proteins harbors a modular F-actin-binding headpiece domain at the C terminus. Headpiece provides one of the two F-actin-binding sites essential for filament bundling. Here, we report the first structure of a functional headpiece domain. The NMR structure of chicken villin headpiece (HP67) reveals two subdomains that share a tightly packed hydrophobic core. The N-terminal subdomain contains bends, turns, and a four-residue alpha-helix as well as a buried histidine residue that imparts a pH-dependent folding. The C-terminal subdomain is composed of three alpha-helices and its folding is pH-independent. Two residues previously implicated in F-actin-binding form a buried salt-bridge between the N and C-terminal subdomains. The rest of the identified actin-binding residues are solvent-exposed and map onto a unique F-actin-binding surface.

• Lim RW, Furukawa R, Eagle S, Cartwright RC, Fechheimer M
• Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein.
• Biochemistry. 1999; 38: 800-12
• Display abstract

• The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.

• Keep NH, Winder SJ, Moores CA, Walke S, Norwood FL, Kendrick-Jones J
• Crystal structure of the actin-binding region of utrophin reveals a head-to-tail dimer.
• Structure Fold Des. 1999; 7: 1539-46
• Display abstract

• BACKGROUND: Utrophin is a large multidomain protein that belongs to a superfamily of actin-binding proteins, which includes dystrophin, alpha-actinin, beta-spectrin, fimbrin, filamin and plectin. All the members of this family contain a common actin-binding region at their N termini and perform a wide variety of roles associated with the actin cytoskeleton. Utrophin is the autosomal homologue of dystrophin, the protein defective in the X-linked Duchenne and Becker muscular dystrophies, and upregulation of utrophin has been suggested as a potential therapy for muscular dystrophy patients. RESULTS: The structure of the actin-binding region of utrophin, consisting of two calponin-homology (CH) domains, has been solved at 3.0 A resolution. It is composed of an antiparallel dimer with each of the monomers being present in an extended dumbell shape and the two CH domains being separated by a long central helix. This extended conformation is in sharp contrast to the compact monomer structure of the N-terminal actin-binding region of fimbrin. CONCLUSIONS: The crystal structure of the actin-binding region of utrophin suggests that these actin-binding domains may be more flexible than was previously thought and that this flexibility may allow domain reorganisation and play a role in the actin-binding mechanism. Thus utrophin could possibly bind to actin in an extended conformation so that the sites previously identified as being important for actin binding may be directly involved in this interaction.

• Larbolette O, Wollscheid B, Schweikert J, Nielsen PJ, Wienands J
• SH3P7 is a cytoskeleton adapter protein and is coupled to signal transduction from lymphocyte antigen receptors.
• Mol Cell Biol. 1999; 19: 1539-46
• Display abstract

• Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

• Sun Y et al.
• Molecular cloning and characterization of human trabeculin-alpha, a giant protein defining a new family of actin-binding proteins.
• J Biol Chem. 1999; 274: 33522-30
• Display abstract

• We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.

• Nakamura T, Takeuchi K, Muraoka S, Takezoe H, Takahashi N, Mori N
• A neurally enriched coronin-like protein, ClipinC, is a novel candidate for an actin cytoskeleton-cortical membrane-linking protein.
• J Biol Chem. 1999; 274: 13322-7
• Display abstract

• Brain-enriched human FC96 protein shows a close sequence similarity to the Dictyostelium actin-binding protein coronin, which has been implicated in cell motility, cytokinesis, and phagocytosis. A phylogenetic tree analysis revealed that FC96 and two other mammalian molecules (p57 and IR10) form a new protein family, the coronin-like protein (Clipin) family; thus hereafter we refer to FC96 as ClipinC. A WD domain and a succeeding alpha-helical region are conserved among coronin and Clipin family members. ClipinC is predominantly expressed in the brain, and discrete areas in the mouse brain were intensely labeled with anti-ClipinC antibodies. ClipinC was also shown to bind directly to F-actin in vitro. Immunocytochemical analysis revealed that ClipinC accumulated at focal adhesions as well as at neurite tips and stress fibers. Furthermore, ClipinC was associated with vinculin, which is a major component of focal contacts. These results indicate that ClipinC is also a component part of the cross-bridge between the actin cytoskeleton and the plasma membrane. These findings and the previously reported function of coronin suggest that ClipinC may play specific roles in the reorganization of neuronal actin structure, a change that has been implicated in both cell motility and growth cone advance.

• Wendt KU, Lenhart A, Schulz GE
• The structure of the membrane protein squalene-hopene cyclase at 2.0 A resolution.
• J Mol Biol. 1999; 286: 175-87
• Display abstract

• Squalene cyclases catalyze a cationic cyclization cascade, which is homologous to a key step in cholesterol biosynthesis. The structure of the enzyme from Alicyclobacillus acidocaldarius has been determined in a new crystal form at 2.0 A resolution (1 A=0.1 nm) and refined to an R-factor of 15.3 % (Rfree=18.7 %). The structure indicates how the initial protonation and the final deprotonation of squalene occur and how the transient carbocations are stabilized. The pathways of the flexible educt squalene from the membrane interior to the active center cavity and of the rigid fused-ring product hopene in the reverse direction are discussed. The enzyme contains eight so-called QW-sequence repeats that fortify the alpha/alpha-barrels by an intricate interaction network. They are unique to the known triterpene cyclases and are presumed to shield these enzymes against the released enthalpy of the highly exergonic catalyzed reaction. The enzyme is a monotopic membrane protein, the membrane-binding interactions of which are described and compared with those of two prostaglandin-H2 synthase isoenzymes, the only other structurally characterized proteins of this type. In the crystals the membrane-binding regions face each other, suggesting a micelle-type detergent structure between them.

• Lappalainen P, Kessels MM, Cope MJ, Drubin DG
• The ADF homology (ADF-H) domain: a highly exploited actin-binding module.
• Mol Biol Cell. 1998; 9: 1951-9

• McGough A
• F-actin-binding proteins.
• Curr Opin Struct Biol. 1998; 8: 166-76
• Display abstract

• The study of proteins that bind filamentous actin (F-actin) is entering an exciting stage as more and more structures are determined. After more than 50 years in which the focus was on muscle proteins, emphasis has recently shifted towards understanding the complex interplay among actin-binding molecules in non-muscle cells. To date, the binding sites for eight classes of filament-binding molecules have been determined by combining low- to intermediate-resolution maps obtained by electron microscopy with atomic structures determined by X-ray crystallography and NMR. Recent results have dramatically accentuated the importance of filament geometry and actin conformation in defining these interactions.

• Fulgenzi G et al.
• Location of the binding site of the mannose-specific lectin comitin on F-actin.
• J Mol Biol. 1998; 284: 1255-63
• Display abstract

• We have used electron microscopy and computer image processing to produce a three-dimensional reconstruction of F-actin filaments decorated with the putative lectin and actin-binding protein comitin. These reconstructions show that comitin binds to F-actin at high radius primarily to actin subdomain 1. This location is distinctly different from the binding site on F-actin for other actin bundling proteins, such as members of the alpha-actinin family, and may result from the positively charged comitin interacting with negatively charged sites near the actin N terminus in subdomain 1. The location of the comitin binding site and its restriction to subdomain 1 on a single actin monomer is consistent with comitin's having a function distinct from other actin-binding proteins and, for example, would enable comitin to link bundled actin filaments to the Golgi.

• Xie Z, Xu W, Davie EW, Chung DW
• Molecular cloning of human ABPL, an actin-binding protein homologue.
• Biochem Biophys Res Commun. 1998; 251: 914-9
• Display abstract

• Based on two partial cDNA sequences, a full-length cDNA sequence for an actin-binding like protein previously named ABPL has been isolated and characterized. ABPL is homologous to the human actin-binding proteins ABP-280 and ABP-278. The predicted sequence for ABPL is 2,705 amino acids in length with a calculated molecular mass of 289 kDa. It contains an amino terminal actin-binding domain followed by 24 tandem repeats of approximately 96 amino acids. Two hinge regions, Hinge I and Hinge II, were located prior to repeats 16 and 24, respectively. An isoform of ABPL lacking Hinge I, with a calculated molecular mass of 286 kDa, was also identified by the reverse transcriptase PCR (RT-PCR) method. A comparison with genomic sequences indicated the isoform resulted from alternative RNA splicing. ABPL has a unique insertion sequence of 82 amino acids in repeat 20 that was not present in the other two homologues and has a tissue distribution that was also different from the other two homologues.

• Benning MM, Wesenberg G, Liu R, Taylor KL, Dunaway-Mariano D, Holden HM
• The three-dimensional structure of 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas sp. Strain CBS-3.
• J Biol Chem. 1998; 273: 33572-9
• Display abstract

• The soil-dwelling microbe, Pseudomonas sp. strain CBS-3, has attracted recent attention due to its ability to survive on 4-chlorobenzoate as its sole carbon source. The biochemical pathway by which this organism converts 4-chlorobenzoate to 4-hydroxybenzoate consists of three enzymes: 4-chlorobenzoyl-CoA ligase, 4-chlorobenzoyl-CoA dehalogenase, and 4-hydroxybenzoyl-CoA thioesterase. Here we describe the three-dimensional structure of the thioesterase determined to 2.0-A resolution. Each subunit of the homotetramer is characterized by a five-stranded anti-parallel beta-sheet and three major alpha-helices. While previous amino acid sequence analyses failed to reveal any similarity between this thioesterase and other known proteins, the results from this study clearly demonstrate that the molecular architecture of 4-hydroxybenzoyl-CoA thioesterase is topologically equivalent to that observed for beta-hydroxydecanoyl thiol ester dehydrase from Escherichia coli. On the basis of the structural similarity between these two enzymes, the active site of the thioesterase has been identified and a catalytic mechanism proposed.

• Bozic D, Engel J, Brancaccio A
• Sequence analysis suggests the presence of an IG-like domain in the N-terminal region of alpha-dystroglycan which was crystallized after mutation of a protease susceptible site (Arg168-->His).
• Matrix Biol. 1998; 17: 495-500
• Display abstract

• Recently, we demonstrated that the N-terminal region of mouse alpha-dystroglycan represents an autonomously folding globular domain, organized into at least two subdomains (Brancaccio et al., Eur. J. Biochem. 246, 166-172, 1997). We have now found a similarity between a part of the alpha-dystroglycan N-terminal sequence (approximately from position 80 to 180) and several protein sequences belonging to the immunoglobulin kappa family. Moreover, we have recombinantly expressed and purified a 31 kDa protein fragment which matches the entire alpha-dystroglycan N-terminal globular domain. To prevent the action of bacterial endogenous proteases and/or thrombin, which cleaves the protein into two fragments at an Arg-Ala trypsin-sensitive site in positions 168-169, we have introduced a single mutation (Arg168-->His), thus making the whole domain more stable and suitable for crystallization. Crystals of this mutant protein were obtained by vapor diffusion using the hanging drop technique, and they diffract to 0.28 nm Bragg spacing.

• Ohtsuka T et al.
• Nexilin: a novel actin filament-binding protein localized at cell-matrix adherens junction.
• J Cell Biol. 1998; 143: 1227-38
• Display abstract

• We isolated two novel actin filament (F-actin)-binding proteins from rat brain and rat 3Y1 fibroblast. They were splicing variants, and we named brain big one b-nexilin and fibroblast small one s-nexilin. b-Nexilin purified from rat brain was a protein of 656 amino acids (aa) with a calculated molecular weight of 78,392, whereas s-nexilin, encoded by the cDNA isolated from rat 3Y1 cells by the reverse transcriptase-PCR method, was a protein of 606 aa with a calculated molecular weight of 71,942. b-Nexilin had two F-actin- binding domains (ABDs) at the NH2-terminal and middle regions, whereas s-nexilin had one ABD at the middle region because 64 aa residues were deleted and 14 aa residues were inserted in the first NH2-terminal ABD of b-nexilin, and thereby the first ABD lost its activity. b- and s-nexilins bound along the sides of F-actin, but only b-nexilin showed F-actin cross-linking activity. b-Nexilin was mainly expressed in brain and testis, whereas s-nexilin was mainly expressed in testis, spleen, and fibroblasts, such as rat 3Y1 and mouse Swiss 3T3 cells, but neither b- nor s-nexilin was detected in liver, kidney, or cultured epithelial cells. An immunofluorescence microscopic study revealed that s-nexilin was colocalized with vinculin, talin, and paxillin at cell- matrix adherens junction (AJ) and focal contacts, but not at cell-cell AJ, in 3Y1 cells. Overexpressed b- and s-nexilins were localized at focal contacts but not at cell-cell AJ. These results indicate that nexilin is a novel F-actin-binding protein localized at cell-matrix AJ.

• Gimona M, Winder SJ
• Single calponin homology domains are not actin-binding domains.
• Curr Biol. 1998; 8: 6745-6745

• McCurdy DW, Kim M
• Molecular cloning of a novel fimbrin-like cDNA from Arabidopsis thaliana.
• Plant Mol Biol. 1998; 36: 23-31
• Display abstract

• Fimbrin is a 68-70 kDa actin-bundling protein in animal cells and lower eukaryotes that participates in diverse morphogenetic processes by cross-linking actin filaments into bundles. Here we report the cloning by degenerate polymerase chain reaction (PCR) of ATFIM1, a 2.3 kb cDNA from Arabidopsis thaliana that codes for a novel 76 kDa fimbrin-like polypeptide (AtFim1). The predicted sequence of AtFim1 shares ca. 40% identity with nonplant fimbrins and contains two tandem repeats, each possessing a 27 amino acid region identified as a putative actin-binding domain in fimbrins and in a larger family of actin cross-linking proteins. Preceding the tandem repeats at the amino terminus of AtFim1 is a single-EF-hand-like domain with moderate homology to calmodulin-like calcium-binding proteins. AtFim1 differs from non-plant fimbrins, however, in that it contains an extended carboxy-terminal tail of ca. 65 amino acids. ATFIM1 is encoded by a single gene, although sequencing of two partial fimbrin-like expressed sequence tag (EST) clones indicates that Arabidopsis contains at least two fimbrin-like proteins. Northern blot analysis and reverse-transcription PCR (RT-PCR) demonstrated that ATFIM1 is expressed in all major organs examined (roots, leaves, stems, flowers and siliques). This is the first report of the cloning of a full length plant gene that encodes a putative actin filament-bundling protein.

• Hanein D et al.
• An atomic model of fimbrin binding to F-actin and its implications for filament crosslinking and regulation.
• Nat Struct Biol. 1998; 5: 787-92
• Display abstract

• Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium.

• Vaduva G, Martin NC, Hopper AK
• Actin-binding verprolin is a polarity development protein required for the morphogenesis and function of the yeast actin cytoskeleton.
• J Cell Biol. 1997; 139: 1821-33
• Display abstract

• Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70- amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.

• Sun S, Footer M, Matsudaira P
• Modification of Cys-837 identifies an actin-binding site in the beta-propeller protein scruin.
• Mol Biol Cell. 1997; 8: 421-30
• Display abstract

• In the acrosomal process of Limulus sperm, the beta-propeller protein scruin cross-links actin into a crystalline bundle. To confirm that scruin has the topology of a beta-propeller protein and to understand how scruin binds actin, we compared the solvent accessibility of cysteine residues in scruin and the acrosomal process by chemical modification with (1,5-IAEDANS). In soluble scruin, the two most reactive cysteines of soluble scruin are C837 and C900, whereas C146, C333, and C683 are moderately reactive. This pattern of reactivity is consistent with the topology of a typical beta-propeller protein; all of the reactive cysteines map to putative loops and turns whereas the unreactive cysteines lie within the predicted interior of the protein. The chemical reactivities of cysteine in the acrosomal process implicate C837 at an actin-binding site. In contrast to soluble scruin, in the acrosomal process, C837 is completely unreactive while the other cysteines become less reactive. Binding studies of chemically modified scruin correlate the extent of modification at C837 with the extent of inhibition of actin binding. Furthermore, peptides corresponding to residues flanking C837 bind actin and narrow a possible actin-binding region to a KQK sequence. On the basis of these studies, our results suggest that an actin-binding site lies in the C-terminal domain of scruin and involves a putative loop defined by C837.

• Blanchard H et al.
• Structure of a calpain Ca(2+)-binding domain reveals a novel EF-hand and Ca(2+)-induced conformational changes.
• Nat Struct Biol. 1997; 4: 532-8
• Display abstract

• The crystal structure of a Ca(2+)-binding domain (dVI) of rat m-calpain has been determined at 2.3 A resolution, both with and without bound Ca2+. The structures reveal a unique fold incorporating five EF-hand motifs per monomer, three of which bind calcium at physiological calcium concentrations, with one showing a novel EF-hand coordination pattern. This investigation gives us a first view of the calcium-induced conformational changes, and consequently an insight into the mechanism of calcium induced activation in calpain. The crystal structures reveal a dVI homodimer which provides a preliminary model for the subunit dimerization in calpain.

• Lin GD et al.
• Crystal structure of calcium bound domain VI of calpain at 1.9 A resolution and its role in enzyme assembly, regulation, and inhibitor binding.
• Nat Struct Biol. 1997; 4: 539-47
• Display abstract

• The three dimensional structure of calcium-bound domain VI of porcine calpain has been determined to 1.9 A resolution. The crystal structure reveals five EF-hands, one more than previously suggested. There are two EF-hand pairs, one pair (EF1-EF2) displays an 'open' conformation and the other (EF3-EF4) a 'closed' conformation. Unusually, a calcium atom is found at the C-terminal end of the calcium binding loop of EF4. With two additional residues in the calcium binding loop, the fifth EF-hand (EF5) is in a 'closed' conformation. EF5 pairs up with the corresponding fifth EF-hand of a non-crystallographically related molecule. Considering the EF5's role in a homodimer formation of domain VI, we suggest a model for the assembly of heterodimeric calpain. The crystal structure of a Ca2+ bound domain VI-inhibitor (PD150606) complex has been refined to 2.1 A resolution. A possible mode for calpain inhibition is discussed.

• Schutt CE, Kreatsoulas C, Page R, Lindberg U
• Plugging into actin's architectonic socket.
• Nat Struct Biol. 1997; 4: 169-72

• Fucini P, Renner C, Herberhold C, Noegel AA, Holak TA
• The repeating segments of the F-actin cross-linking gelation factor (ABP-120) have an immunoglobulin-like fold.
• Nat Struct Biol. 1997; 4: 223-30
• Display abstract

• The 120,000 M(r) gelation factor and alpha-actinin are among the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are rod-shaped homodimers. Each monomer chain is comprised of an actin-binding domain and a rod domain. The rod domain of the gelation factor consists of six 100-residue repetitive segments with high internal homology. We have now determined the three-dimensional structure of segment 4 of the rod domain of the gelation factor from D. discoideum using NMR spectroscopy. The segment consists of seven beta-sheets arranged in an immunoglobulin-like (Ig) fold. This is completely different from the alpha-actinin rod domain which consists of four spectrin-like alpha-helical segments. The gelation factor is the first example of an Ig-fold found in an actin-binding protein. Two highly homologous actin-binding proteins from human with similar sequences to the gelation factor, filamin and a 280,000 M(r) actin-binding protein (ABP-280), share conserved residues that form the core of the gelation factor repetitive segment structure. Thus, the segment 4 structure should be common to this subfamily of the spectrin superfamily. The structure of segment 4 together with previously published electron microscopy data, provide an explanation for the dimerization of the whole gelation factor molecule.

• Burtnick LD et al.
• The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.
• Cell. 1997; 90: 661-70
• Display abstract

• The structure of gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).

• Krause KH, Michalak M
• Calreticulin.
• Cell. 1997; 88: 439-43

• Mahoney NM, Janmey PA, Almo SC
• Structure of the profilin-poly-L-proline complex involved in morphogenesis and cytoskeletal regulation.
• Nat Struct Biol. 1997; 4: 953-60
• Display abstract

• Profilin, a ubiquitous low molecular weight (13,000-15,000 M(r)) actin binding protein, regulates the formation of F-actin structures in vivo, and is localized to specific cellular regions through interaction with proline-rich sequences. Here we report the 2.2 A X-ray structure of the complex between human platelet profilin (HPP) and a decamer of L-proline (L-Pro10). The L-Pro10 peptide adopts a left-handed type II poly-L-proline helix (PPII) and binds to a highly conserved patch of aromatic amino acids on the surface of profilin. The peptide and actin binding sites reside on orthogonal surfaces, and L-Pro10 binding does not result in a conformational rearrangement of HPP. This structure suggests a mechanism for the localization of profilin and its actin-related activities to sites of actin filament assembly in vivo.

• Suphioglu C, Ferreira F, Knox RB
• Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen.
• FEBS Lett. 1997; 402: 167-72
• Display abstract

• A cDNA coding for a newly identified Bermuda grass pollen allergen, Cyn d 7, with significant sequence similarity to Ca2+-binding proteins, was isolated from a cDNA expression library using serum IgE from an allergic individual. The deduced amino acid sequence of Cyn d 7 contained two typical Ca2+-binding sites (EF hand domains). Depletion of Ca2+ with EGTA led to a loss of IgE-binding capacity of rCyn d 7. A synthetic peptide based on domain II showed high IgE reactivity. Cyn d 7 therefore represents a grass pollen allergen that belongs to a novel class of Ca2+-binding proteins.

• Feinberg J, Mery J, Heitz F, Benyamin Y, Roustan C
• Conformational and functional studies of three gelsolin subdomain-1 synthetic peptides and their implication in actin polymerization.
• Biopolymers. 1997; 41: 647-55
• Display abstract

• Gelsolin, a calcium and inositol phospholipid-sensitive protein, regulates actin filament length. Its activity is complex (capping, severing, etc.) and is supported by several functional domains. The N-terminal domain alone (S1), in particular, is able to impede actin polymerization. Our investigations were attempted to precise this inhibitory process by using synthetic peptides as models mimicking gelsolin S1 activity. Three peptides issued from S1 and located in gelsolin-actin interfaces were synthesized. The peptides (15-28, 42-55, and 96-114 sequences) were tested for their conformational and actin binding properties. Although the three peptides interact well with actin, only peptide 42-55 affects actin polymerization. A detailed kinetic study shows that the latter peptide essentially inhibits the nucleation step during actin polymerization. In conclusion, the present work shows that the binding of a synthetic peptide to a small sequence located outside the actin-actin interface is essential in the actin polymerization process.

• Tang JX, Szymanski PT, Janmey PA, Tao T
• Electrostatic effects of smooth muscle calponin on actin assembly.
• Eur J Biochem. 1997; 247: 432-40
• Display abstract

• The contribution of electrostatic interactions to the effects of chicken gizzard calponin on the kinetics of actin polymerization and the bundling of F-actin were characterized by a combination of fluorescence, light-scattering, co-sedimentation, and electron-microscopic methods. Stoichiometric amounts of calponin accelerate actin polymerization in low-ionic-strength solutions, but this effect is diminished at [KCI] = 150 mM. At low ionic strengths, micromolar concentrations of calponin induce the formation of large bundles of actin filaments, and lower concentrations of calponin quench the fluorescence of pyrene-labeled F-actin. The latter effect is related to binding of calponin to F-actin rather than to bundling of the filaments. The concentration of calponin required to bundle a fixed concentration of actin filaments increases with increasing ionic strength, as the average diameter of the bundles decreases. Millimolar concentrations of ATP, GTP or ITP are equally efficient at dispersing actin bundles to single filaments or smaller aggregates, even though a significant fraction of calponin remains bound to F-actin. Our findings show that the binding of calponin to actin is determined at least in part by electrostatic interactions, and that the polycationic nature of calponin is primarily responsible for the formation of F-actin bundles via its ability to reduce the electrostatic repulsion between the negatively charged actin filaments.

• Bashour AM, Fullerton AT, Hart MJ, Bloom GS
• IQGAP1, a Rac- and Cdc42-binding protein, directly binds and cross-links microfilaments.
• J Cell Biol. 1997; 137: 1555-66
• Display abstract

• Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two approximately 190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D-sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.

• Frazier JA, Field CM
• Actin cytoskeleton: are FH proteins local organizers?
• Curr Biol. 1997; 7: 4147-4147
• Display abstract

• The FH proteins, defined by the presence of 'formin homology' regions, are important for a number of actin-dependent processes, including polarized cell growth and cytokinesis. They are large, probably multi-domain, proteins and their function may be in part mediated by an interaction with profilin.

• Choi HK, Lu G, Lee S, Wengler G, Rossmann MG
• Structure of Semliki Forest virus core protein.
• Proteins. 1997; 27: 345-59
• Display abstract

• Alphaviruses are enveloped, insect-borne viruses, which contains a positive-sense RNA genome. The protein capsid is surrounded by a lipid membrane, which is penetrated by glycoprotein spikes. The structure of the Sindbis virus (SINV) (the type virus) core protein (SCP) was previously determined and found to have a chymotrypsin-like structure. SCP is a serine proteinase which cleaves itself from a polyprotein. Semliki Forest virus (SFV) is among the most distantly related alphaviruses to SINV. Similar to SCP, autocatalysis is inhibited in SFCP after cleavage of the polyprotein by leaving the carboxy-terminal tryptophan in the specificity pocket. The structures of two different crystal forms (I and II) of SFV core protein (SFCP) have been determined to 3.0 A and 3.3 A resolution, respectively. The SFCP monomer backbone structure is very similar to that of SCP. The dimeric association between monomers, A and B, found in two different crystal forms of SCP is also present in both crystal forms of SFCP. However, a third monomer, C, occurs in SFCP crystal form I. While monomers A and B make a tail-to-tail dimer contact, monomers B and C make a head-to-head dimer contact. A hydrophobic pocket on the surface of the capsid protein, the proposed site of binding of the E2 glycoprotein, has large conformational differences with respect to SCP and, in contrast to SCP, is found devoid of bound peptide. In particular, Tyr184 is pointing out of the hydrophobic pocket in SFCP, whereas the equivalent tyrosine in SCP is pointing into the pocket. The conformation of Tyr184, found in SFCP, is consistent with its availability for iodination, as observed in the homologous SINV cores. This suggests, by comparison with SCP, that E2 binding to cores causes major conformational changes, including the burial of Tyr184, which would stabilize the intact virus on budding from an infected cell. The head-to-tail contacts found in the pentameric and hexameric associations within the virion utilize in the same monomer surface regions as found in the crystalline dimer interfaces.

• Ervasti JM, Rybakova IN, Amann KJ
• A multiple site, side binding model for the interaction of dystrophin with F-actin.
• Soc Gen Physiol Ser. 1997; 52: 31-44

• Whittaker M, Milligan RA
• Conformational changes due to calcium-induced calmodulin dissociation in brush border myosin I-decorated F-actin revealed by cryoelectron microscopy and image analysis.
• J Mol Biol. 1997; 269: 548-57
• Display abstract

• Brush border myosin I (BBMI) is a single-headed molecular motor. Its catalytic domain exhibits extensive sequence homology to the catalytic domain of myosin II, while its tail lacks the coiled-coil nature of myosin II. The BBMI tail domain contains at least three IQ motifs and binds calmodulin. Addition of calcium removes one of these calmodulin light chains, with effects on ATPase activity and motility in in vitro assays. Using the techniques of cryoelectron microscopy and helical image analysis we have calculated three-dimensional (3D) maps of BBMI-decorated actin filaments prepared in the presence and absence of calcium. The 3D maps describe a BBMI catalytic domain that is strikingly similar to the catalytic domain of myosin II subfragment 1 (S1), with the exception of a short amino-terminal region of the heavy chain, which is absent from BBMI. The tail domains of BBMI and S1 are highly divergent in structure, continuing on from their respective motor domains with very different geometries. Addition of calcium to BBMI, and the concomitant loss of a calmodulin light chain, results in an extensive reorganization of mass in the tail domain.

• Terasaki AG, Ohnuma M, Mabuchi I
• Identification of actin-binding proteins from sea urchin eggs by F-actin affinity column chromatography.
• J Biochem (Tokyo). 1997; 122: 226-36
• Display abstract

• Novel F-actin binding proteins of sea urchin eggs were searched for in order to study regulation of the actin cytoskeleton during fertilization and cell division. An extract of unfertilized eggs was analyzed by F-actin column chromatography. Several previously characterized F-actin-modulating proteins such as spectrin, myosin, and fascin bound to the column. The eluates from the column also contained proteins having apparent molecular weights of 225K, 150K, 70K, 60K, 45K, 40K, 38K, 36K, 34K, 20K, and 15K, which were thought to be novel cytoskeletal proteins judging from their molecular weights and non-reactivity to antibodies against previously characterized F-actin-modulating proteins. Most of the proteins in the F-actin column eluates co-sedimented with F-actin. Partial amino acid sequences of the peptides derived from the 45K and 40K proteins showed that these proteins are homologous to Arp3 and Arp2 subfamilies of actin-related proteins, respectively. The 150K protein seemed to be an unconventional myosin, that belongs to myosin VI subfamily. Amino acid sequences of two fragments from the 60K protein showed homology to that of coronin. The 150K protein was localized by immunofluorescence microscopy to the cleavage furrows in both whole cell sample and isolated cortex of dividing eggs. The 70K protein was uniformly localized in the cortical layer in the whole egg, but weak staining of the cleavage furrow region with the antiserum was observed in the isolated cortex. The 60K protein was localized to both the bulk cortical layer and the cleavage furrow, but the modes of localization were different.

• Shih CL, Chen MJ, Linse K, Wang K
• Molecular contacts between nebulin and actin: cross-linking of nebulin modules to the N-terminus of actin.
• Biochemistry. 1997; 36: 1814-25
• Display abstract

• Nebulin, a giant actin binding protein, coextends with actin and is thought to form a composite thin filament in the skeletal muscle sarcomere. To understand the molecular interactions between nebulin and actin, we have applied chemical cross-linking techniques to define molecular contacts between actin and ND8, a two-module nebulin fragment that promotes actin polymerization and inhibits depolymerization by binding to both G- and F-actin. The formation of a 1:1 complex with a dissociation constant of 4.9 microM between ND8 and G-actin was demonstrated by fluorescence titration of dansyl-ND8 with G-actin. Treatment with a zero-length cross-linker, l-ethyl-3-[3-(dimethylamino) propyl]carbodiimide (EDC), cross-linked the ND8-G-actin complex covalently without impairing actin's ability to polymerize. End-labeling Western blot and sequence and mass analyses of purified conjugated peptides revealed the cross-linking between lysine 5 of ND8 and the two N-terminal acidic residues of G-actin. Similarly, we have shown by end-labeling that cross-linking of ND8 to F-actin occurred at the N-terminus of actin protomer. The binding of nebulin to the N-terminus of actin is likely to be significant in its ability to affect actin polymerization. Furthermore, the association of nebulin modules with the actin N-terminus in subdomain 1 supports the hypothesis that nebulin wraps around the outer edges of actin filaments where Sl, tropomyosin, and several actin binding proteins are known to interact.

• Orlova A, Chen X, Rubenstein PA, Egelman EH
• Modulation of yeast F-actin structure by a mutation in the nucleotide-binding cleft.
• J Mol Biol. 1997; 271: 235-43
• Display abstract

• Although the actin sequence is very highly conserved across evolution, tissue-specific expression of different isoforms in high eukaryotes suggests that different isoforms carry out different functions. However, little information exists about either the differences in filaments made from different actins or the effects on filament structure caused by the various mutations in actin that have been introduced to gain insight into actin function. Using electron microscopy and three-dimensional reconstruction, we have studied the differences in the filaments made by yeast and rabbit skeletal muscle actin, two proteins with 88% homologous sequences, and we have assessed the changes in filament structure caused by the introduction of the S14A mutation into yeast actin. Elimination of the S14 hydroxyl group, assumed to bind to the gamma-phosphate of actin-bound ATP, results in a 40 to 60-fold decrease in actin's affinity for ATP. We show that yeast actin displays less extensive contacts between the two long-pitch helical strands than does muscle actin, and displays the large cooperativity within filaments previously observed for muscle actin. Finally, we demonstrate that the S14A mutation narrows the cleft between the two lobes of the actin subunit and strengthens the inter-strand connections in F-actin.

• Prassler J, Stocker S, Marriott G, Heidecker M, Kellermann J, Gerisch G
• Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.
• Mol Biol Cell. 1997; 8: 83-95
• Display abstract

• A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca(2+)-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the alpha-actinin/beta-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to beta/gamma-actin from bovine spleen but not to alpha-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of protein-protein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.

• Goldsmith SC, Pokala N, Matsudaira P, Almo SC
• Crystallization and preliminary crystallographic analysis of the N-terminal actin binding domain of human fimbrin.
• Proteins. 1997; 28: 452-3
• Display abstract

• We have crystallized the N-terminal actin binding domain (ABD1) of human fimbrin, a representative member of the largest class of actin crosslinking proteins. Diffraction from these crystals is consistent with the orthorhombic space group P2(1)2(1)2(1) (a = 50.03 A, b = 61.24 A, c = 102.30 A). These crystals contain one molecule in the asymmetric unit and diffract to at least 1.9 A resolution. The crystal structure of ABD1 will be the first structure of an actin crosslinking domain.

• Mounier N, Sparrow JC
• Structural comparisons of muscle and nonmuscle actins give insights into the evolution of their functional differences.
• J Mol Evol. 1997; 44: 89-97
• Display abstract

• Actin is a highly conserved protein although many isoforms exist. In vertebrates and insects the different actin isoforms can be grouped by their amino acid sequence and tissue-specific gene expression into muscle and nonmuscle actins, suggesting that the different actins may have a functional significance. We ask here whether atomic models for G- and F-actins may help to explain this functional diversity. Using a molecular graphics program we have mapped the few amino acids that differ between isoactins. A small number of residues specific for muscle actins are buried in internal positions and some present a remarkable organization. Within the molecule, the replacements observed between muscle and nonmuscle actins are often accompanied by compensatory changes. The others are dispersed on the protein surface, except for a cluster located at the N-terminus which protrudes outward. Only a few of these residues specific for muscle actins are present in known ligand binding sites except the N-terminus, which has a sequence specific for each isoactin and is directly implicated in the binding to myosin. When we simulated the replacements of side chains of residues specific for muscle actins to those specific for nonmuscle actins, the N-terminus appears to be less compact and more flexible in nonmuscle actins. This would represent the first conformational grounds for proposing that muscle and nonmuscle actins may be functionally distinguishable. The rest of the molecule is very similar or identical in all the actins, except for a possible higher internal flexibility in muscle actins. We propose that muscle actin genes have evolved from genes of nonmuscle actins by substitutions leading to some conformational changes in the protruding N-terminus and the internal dynamics of the main body of the protein.

• Thorn KS et al.
• The crystal structure of a major allergen from plants.
• Structure. 1997; 5: 19-32
• Display abstract

• BACKGROUND: Profilins are small eukaryotic proteins involved in modulating the assembly of actin microfilaments in the cytoplasm. They are able to bind both phosphatidylinositol-4,5-bisphosphate and poly-L-proline (PLP) and thus play a critical role in signaling pathways. Plant profilins are of interest because immunological cross-reactivity between pollen and human profilin may be the cause of hay fever and broad allergies to pollens. RESULTS: The determination of the Arabidopsis thaliana profilin isoform I structure, using multiwavelength anomalous diffraction (MAD) to obtain structure-factor phases, is reported here. The structure of Arabidopsis profilin is similar to that of previously determined profilin structures. Conserved amino acid residues in profilins from plants, mammals, and lower eukaryotes are critically important in dictating the geometry of the PLP-binding site and the overall polypeptide fold. The main feature distinguishing plant profilins from other profilins is a solvent-filled pocket located in the most variable region of the fold. CONCLUSIONS: Comparison of the structures of SH3 domains with those of profilins from three distinct sources suggests that the mode of PLP binding may be similar. A comparison of three profilin structures from different families reveals only partial conservation of the actin-binding surface. The proximity of the semi-conserved actin-binding site and the binding pocket characteristic of plant profilins suggests that epitopes encompassing both features are responsible for the cross-reactivity of antibodies between human and plant profilins thought to be responsible for type I allergies.

• Maurer P, Hohenester E, Engel J
• Extracellular calcium-binding proteins.
• Curr Opin Cell Biol. 1996; 8: 609-17
• Display abstract

• Point mutations in Ca2+-binding sites of extracellular matrix proteins have been identified as the cause of human disorders such as Marfansyndrome and pseudoachondroplasia. Although the modes of Ca2+ binding and the effects of point mutations are not yet understood in these two cases, new insight was recently gained by X-ray and NMR structure determinations of several other extracellular proteins; these studies revealed a diversity of functions of Ca2+ ions. Ca2+ may induce a profound conformational change within a single domain, may bridge adjacent domains and thus direct the relative domain orientation and supramolecular structure, or may be involved in carbohydrate and membrane binding.

• Sandalova TP
• [Modeling the spatial structure of obelin--a calcium-activated photoprotein from the hydroid Obelia longissima]
• Mol Biol (Mosk). 1996; 30: 621-30

• Rybakova IN, Amann KJ, Ervasti JM
• A new model for the interaction of dystrophin with F-actin.
• J Cell Biol. 1996; 135: 661-72
• Display abstract

• The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super-family of proteins. However, we failed to observe dystrophin-glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin-glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F-actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins.

• Markus MA, Dayie KT, Matsudaira P, Wagner G
• Local mobility within villin 14T probed via heteronuclear relaxation measurements and a reduced spectral density mapping.
• Biochemistry. 1996; 35: 1722-32
• Display abstract

• Villin 14T, a representative domain from the actin severing and bundling protein villin, binds calcium ions and actin monomers. To begin to understand the contributions of mobility to the villin-calcium and villin-actin interactions, relaxation rates for magnetization involving the amide nitrogens and protons have been measured for 15N-labeled villin 14T in solution. Although we have measured the complete set of rates required for a full spectral density map, difficulties in the accurate measurement of relaxation rates for antiphase coherence and two-spin order led us to consider a reduced mapping formalism. From the reduced spectral density map, a model-free analysis, or directly from the measured Nx,y relaxation rates, local variations in mobility along the backbone of villin 14T have been revealed. Fast motions are evident not only at the amino and carboxyl termini but also in the turn between strands beta 4 and beta 5 of the central beta-sheet and in the turn between helix alpha 3 and strand beta 7. Slower motions are suggested for the turn between strands beta 2 and beta 3. Motions on the microsecond to millisecond time scale have been probed directly by examining the dependence of the proton transverse relaxation rate on the spin-locking field strength. Leu11 shows a strong dependence on field strength, implying conformational exchange with a time constant of 125 +/- 69 microseconds. The backbone at the actin-binding interface appears to be rather rigid.

• Liu SH, Gottsch JD
• Amino acid sequence of an immunogenic corneal stromal protein.
• Invest Ophthalmol Vis Sci. 1996; 37: 944-8
• Display abstract

• PURPOSE: A unique cornea-associated antigen (CO-Ag) has been purified previously from stromal extracts. The protein is the target for autoantibodies in patients with Mooren's ulcer. In this study, the amino acid sequence of CO-Ag was analyzed and the structure-function properties of CO-Ag was determined. METHODS: Purified CO-Ag was subjected to N-terminal sequencing by automated Edman degradation. Binding of calcium (Ca2+) to CO-Ag was measured by a direct (45)Ca2+ -binding assay. RESULTS: The complete amino acid sequence of CO-Ag has been determined. The protein contains 70 amino acids in a single chain and lacks cysteine, tryptophan, and methionine residues. A computerized data base search of protein and nucleic acid sequences revealed strong homology to the Ca2+ -binding proteins of the S-100 family. The sequence of CO-Ag shows a high homology with calgranulin C (CaG-C) previously purified from pig granulocytes. The functional Ca2+ -binding sites of CO-Ag and CaG-C were different based on homology with known Ca2+ -binding domains and their Ca2+ -binding properties. There are three amino acid substitutions in the N-terminal Ca2+ -binding domain. Differences were functionally conserved and compatible, with minimum single-base changes in the codon structures. The greatest difference was located in the C-terminal Ca2+ -binding domain. Five consecutive amino acid changes from D63-K-K-G-A67 in CO-Ag to M63-Q-D-E-Q67 occurred in CaG-C. These differences alter the structure of CO-Ag, which no longer can bind Ca2+ ions. The existence of this nonfunctional Ca2+ -binding site was corroborated by its Ca2+ -binding properties. The number of Ca2+ -binding sites for the CO-Ag sub-unit is approximately half that of the CaG-C monomer, although these two proteins have a similar low binding constant of approximately 2 x 10(-4) M. CONCLUSIONS: These results suggest that CO-Ag is a new member of the Ca2+ -binding protein of the S-100 family heretofore undescribed in the cornea. Sequence data provide an important framework to search for sequence similarity with microbial proteins as possible substrates for molecular mimicry and for the identification of possible pathogenic epitopes in CO-Ag.

• Wedaman KP, Meyer DW, Rashid DJ, Cole DG, Scholey JM
• Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex.
• J Cell Biol. 1996; 132: 371-80
• Display abstract

• The heterotrimeric kinesin-II holoenzyme purified from sea urchin (Strongylocentrotus purpuratus) eggs is assembled from two heterodimerized kinesin-related motor subunits of known sequence, together with a third, previously uncharacterized 115-kD subunit, SpKAP115. Using monospecific anti-SpKAP115 antibodies we have accomplished the molecular cloning and sequencing of the SpKAP115 subunit. The deduced sequence predicts a globular 95-kD non-motor "accessory" polypeptide rich in alpha-helical segments that are generally not predicted to form coiled coils. Electron microscopy of individual rotary shadowed kinesin-II holoenzymes also suggests that SpKAP115 is globular, with a somewhat asymmetric morphology. Moreover, the SpKAP115 subunit lies at one end of the 51-nm-long kinesin-II complex, being separated from the two presumptive motor domains by a approximately 26-nm-long rod, in a manner similar to the light chains (KLCs) of kinesin itself. This indicates that SpKAP115 and the KLCs may have analogous functions, yet SpKAP115 does not display significant sequence similarity with the KLCs. The results show that kinesin and kinesin-II are assembled from highly divergent accessory polypeptides together with kinesin related motor subunits (KRPs) containing conserved motor domains linked to divergent tails. Despite the lack of sequence conservation outside the motor domains, there is striking conservation of the ultrastructure of the kinesin and kinesin-II holoenzymes.

• Bertrand JA, Pignol D, Bernard JP, Verdier JM, Dagorn JC, Fontecilla-Camps JC
• Crystal structure of human lithostathine, the pancreatic inhibitor of stone formation.
• EMBO J. 1996; 15: 2678-84
• Display abstract

• Human lithostathine (HLIT) is a pancreatic glycoprotein which inhibits the growth and nucleation of calcium carbonate crystals. The crystal structure of the monomeric 17 kDa HLIT, determined to a resolution of 1.55 angstroms, was refined to a crystallographic R-factor of 18.6%. Structural comparison with the carbohydrate-recognition domains of rat mannose-binding protein and E-selectin indicates that the C-terminal domain of HLIT shares a common architecture with the C-type lectins. Nevertheless, HLIT does not bind carbohydrate nor does it contain the characteristic calcium-binding sites of the C-type lectins. In consequence, HLIT represents the first structurally characterized member of this superfamily which is not a lectin. Analysis of the charge distribution and calculation of its dipole moment reveal that HLIT is a strongly polarized molecule. Eight acidic residues which are separated by regular 6 angstrom spacings form a unique and continuous patch on the molecular surface. This arrangement coincides with the distribution of calcium ions on certain planes of the calcium carbonate crystal; the dipole moment of HLIT may play a role in orienting the protein on the crystal surface prior to the more specific interactions of the acidic residues.

• Wiech H et al.
• Characterization of green alga, yeast, and human centrins. Specific subdomain features determine functional diversity.
• J Biol Chem. 1996; 271: 22453-61
• Display abstract

• Centrins are a subfamily within the superfamily of Ca2+-modulated proteins that play a fundamental role in centrosome duplication and contraction of centrin-based fiber systems. We examined the individual molecular properties of yeast, green alga, and human centrins. Circular dichroism spectroscopy revealed a divergent influence of Ca2+ binding on the alpha-helical content of these proteins. Ca2+-free centrins were elongated in shape as determined by size exclusion chromatography. The presence of Ca2+ and binding peptide resulted in more spherical shaped centrins. In contrast to yeast calmodulin, centrins formed multimers in the Ca2+-bound state. This oligomerization was significantly reduced in the absence of Ca2+ and in the presence of binding peptide. The Ca2+-dependent polymerization of the green alga Scherffelia dubia centrin (SdCen) resulted in a filamentous network. This molecular property was mainly dependent on the amino-terminal subdomain and the peptide-binding site of SdCen. Finally, we analyzed whether SdCen and Cdc31p-SdCen hybrid proteins functionally substitute for the Saccharomyces cerevisiae centrin Cdc31p. Only hybrid proteins containing the amino-terminal subdomain or the third EF-hand of SdCen and the other subdomains from Cdc31p were functional in vivo.

• Tormo J et al.
• Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.
• EMBO J. 1996; 15: 5739-51
• Display abstract

• The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.

• Van Troys M, Dewitte D, Goethals M, Vandekerckhove J, Ampe C
• Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments 1 and 2 of gelsolin evolved to divergent actin binding functions?
• FEBS Lett. 1996; 397: 191-6
• Display abstract

• Gelsolin is built up of six homologous segments that perform different functions on actin. Segments 1 and 2, which are suggested to be highly similar in their overall folds, bind monomeric and filamentous actin respectively. A long alpha-helix in segment 1 forms the major contact site of this segment with actin. We show that sequence 197-226 of segment 2, equivalent to the region around the actin binding helix in segment 1, contains F-actin binding activity. Consequently, positionally similar parts of segment 1 and 2 are implicated in the actin contact and solvent exposed residues in these parts must have evolved differentially to meet their different actin binding properties.

• Van Troys M, Dewitte D, Goethals M, Carlier MF, Vandekerckhove J, Ampe C
• The actin binding site of thymosin beta 4 mapped by mutational analysis.
• EMBO J. 1996; 15: 201-10
• Display abstract

• We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.

• Feinberg J, Mery J, Heitz F, Benyamin Y, Roustan C
• Correlations between biological activity and structural properties for two short homologous sequences in thymosin beta4 and gelsolin.
• Int J Pept Protein Res. 1996; 47: 62-9
• Display abstract

• Gelsolin and thymosin beta4 appear to be two important actin-associated proteins involved in the regulation of actin polymerization. It has been widely demonstrated that thymosin is the major cellular actin-sequestering factor shifting the polymerization equilibrium of actin towards a monomeric state. At the same time gelsolin, a Ca2+ and inositol phosphate sensitive protein, regulates actin filament length. The interactions of these two proteins with actin are rather complex and require the participation of several complementary peptide sequences. We have identified a common motif, (I, V)EKFD, in the two proteins in the functional sequences so far examined. Gelsolin- and thymosin beta4-related peptides including the common motif were synthesized and their structural and functional properties studied. These two sequences exert a major inhibitory effect on salt-induced actin polymerization. We used circular dichroism and Fourier-transform infrared spectroscopy to show that the two synthetic peptides present some secondary structure in solution. As far as the peptide derived from the thymosin sequence was concerned, alpha-helical structure was induced by trifluoroethanol as observed with the full-length molecule. These experiments underscore the importance of the conformational state of peptide fragments in their biological activities. ELISA and fluorescence measurements have been used to identify the binding regions of these fragments to a C-terminal region (subdomain 1) of the actin sequence. Our results also emphasize the relationship between the propensity of small sequences to form secondary structures and their propensity for biological activity as related to actin interaction and inhibition of actin polymerization.

• Hitomi J, Maruyama K, Kikuchi Y, Nagasaki K, Yamaguchi K
• Characterization of a new calcium-binding protein abundant in amniotic fluid, CAAF2, which is produced by fetal epidermal keratinocytes during embryogenesis.
• Biochem Biophys Res Commun. 1996; 228: 757-63
• Display abstract

• By using a 45Ca2+ overlay technique, we found two calcium-binding proteins (CaBPs) abundant in bovine amniotic fluid, and named them CAAF1 (calcium-binding protein in amniotic fluid-1) and CAAF2. CAAF1 was identified as a novel S100 protein produced by fetal epidermal keratinocytes, squamous epithelial cells and polymorphonuclear leukocytes. Here, we report the primary structure and the tissue distribution of bovine CAAF2. CAAF2 is a newly isolated protein with the two EF hand motifs peculiar to S100 proteins, and 29.7% homology to CAAF1. CAAF2 is 63.4% homologous to human psoriasin, a S100 protein up-regulated in psoriatic epidermis. Northern blot analysis demonstrates that CAAF2 is unique in that it is exclusively expressed in the epidermis. Our results suggest that the majority of CaBPs in amniotic fluid consist of two distinct S100 proteins, CAAF1 and CAAF2, which are produced by fetal epidermal keratinocytes.

• Faix J et al.
• Cortexillins, major determinants of cell shape and size, are actin-bundling proteins with a parallel coiled-coil tail.
• Cell. 1996; 86: 631-42
• Display abstract

• Cortexillins I and II of D. discoideum constitute a novel subfamily of proteins with actin-binding sites of the alpha-actinin/spectrin type. The C-terminal halves of these dimeric proteins contain a heptad repeat domain by which the two subunits are joined to form a two-stranded, parallel coiled coil, giving rise to a 19 nm tail. The N-terminal domains that encompass a consensus actin-binding sequence are folded into globular heads. Cortexillin-linked actin filaments form preferentially anti-parallel bundles that associate into meshworks. Both cortexillins are enriched in the cortex of locomoting cells, primarily at the anterior and posterior ends. Elimination of the two isoforms by gene disruption gives rise to large, flattened cells with rugged boundaries, portions of which are often connected by thin cytoplasmic bridges. The double-mutant cells are multinucleate owing to a severe impairment of cytokinesis.

• Janssen KP et al.
• Viscoelastic properties of F-actin solutions in the presence of normal and mutated actin-binding proteins.
• Arch Biochem Biophys. 1996; 325: 183-9
• Display abstract

• A minimal level of viscoelasticity in the cytoskeleton is an essential prerequisite of cellular motility. To determine the influence of the F-actin crosslinking proteins alpha-actinin and actin-binding protein (ABP)120 gelation factor from Dictyostelium discoideum on the properties of actin gels we used a torsion pendulum to measure directly viscoelastic changes of the filamentous networks. Using the capping proteins severin and DS151 to control actin filament length, both crosslinkers were found to increase the elasticity and the viscosity of F-actin solutions. In the case of alpha-actinin, this activity was completely blocked by micromolar concentrations of Ca2+. The inhibitory functions of the two EF hands of alpha-actinin were further investigated by introducing point mutations into either one or both of the Ca(2+)-binding regions. Mutations in the Ca(2+)-coordinating amino acid residues in the first or in both EF hands left the dynamic storage and loss moduli of the F-actin solution unaltered, independent of the Ca2+ concentration. However, alpha-actinin mutated in the second EF hand increased the viscoelasticity of actin gels like the wild-type protein in the absence of Fa2+. The ABP120 gelation factor exhibited only negligible differences to alpha-actinin in viscometry measurements, whereas its impact on the ratio G"/G' (the ratio of energy lost compared to elastically stored during a deformation) of F-actin solutions was clearly smaller than that of alpha-actinin. We conclude from these data that: (i) a torsion pendulum is an excellent tool to determine small changes of activity in normal and mutated actin-binding proteins, (ii) the first EF hand of alpha-actinin is crucial for its crosslinking function, and (iii) the viscoelastic properties of F-actin gels crosslinked by either alpha-actinin or the ABP120 gelation factor are different.

• Ohmiya Y, Hirano T
• Shining the light: the mechanism of the bioluminescence reaction of calcium-binding photoproteins.
• Chem Biol. 1996; 3: 337-47
• Display abstract

• The Ca2+-binding photoproteins from jellyfish have the unique ability to emit blue light in the presence of calcium ions but without molecular oxygen or any other cofactor. Although there is no crystallographic data on the structure of the photoprotein complex, structure-activity studies have elucidated many features of the complex and many aspects of the mechanism of the bioluminescence reaction.

• el-Mezgueldi M
• Calponin.
• Int J Biochem Cell Biol. 1996; 28: 1185-9
• Display abstract

• Calponin is a troponin-T like protein purified from chicken gizzard smooth muscle. It binds to actin, myosin, Ca(2+)-binding proteins and tropomyosin and inhibits the actomyosin ATPase as well as the movement of actin filaments over myosin in vitro. These properties have led to the proposal that calponin may be involved in the Ca(2+)-dependent regulation of actin-myosin interaction and consequently of smooth muscle contraction. Calponin is localized in both the contractile and the cytoskeletal parts of the smooth muscle cell and may have a structural function in smooth muscle cells. It may also regulate the pool of free actin available for cytoskeleton organization. In vitro calponin function is modulated by its interaction with a Ca(2+)-binding protein and/or by its phosphorylation. This suggests that calponin may play an important role in signal transduction from the membrane receptor to the contractile proteins in smooth muscle.

• Tang JX, Janmey PA
• The polyelectrolyte nature of F-actin and the mechanism of actin bundle formation.
• J Biol Chem. 1996; 271: 8556-63
• Display abstract

• Polymerized (F-)actin is induced to form bundles by a number of polycations including divalent metal ions, Co(NH3)63+, and basic polypeptides. The general features of bundle formation are largely independent of the specific structure of the bundling agent used. A threshold concentration of polycation is required to form lateral aggregates of actin filaments. The threshold concentration varies strongly with the valence of the cation and increases with the ionic strength of the solution. Polyanions such as nucleoside phosphates or oligomers of acidic amino acids disaggregate actin bundles into single filaments. These features are similar to the phenomenon of DNA condensation and can be explained analogously by polyelectrolyte theories. Similar results were found when F-actin was bundled by the peptide corresponding to the actin binding site of myristoylated alanine-rich protein kinase C substrate protein (MARCKS) or by smooth muscle calponin, suggesting that a broad class of actin bundling factors may function in a common manner. Physiologic concentrations of both small ions and large proteins can induce actin interfilament association independent of a requirement for specific binding sites.

• Byers TJ, Beggs AH, McNally EM, Kunkel LM
• Novel actin crosslinker superfamily member identified by a two step degenerate PCR procedure.
• FEBS Lett. 1995; 368: 500-4
• Display abstract

• Actin-crosslinking proteins link F-actin into the bundles and networks that constitute the cytoskeleton. Dystrophin, beta-spectrin, alpha-actinin, ABP-120, ABP-280, and fimbrin share homologous actin-binding domains and comprise an actin crosslinker superfamily. We have identified a novel member of this superfamily (ACF7) using a degenerate primer-mediated PCR strategy that was optimized to resolve less-abundant superfamily sequences. The ACF7 gene is on human chromosome 1 and hybridizes to high molecular weight bands on northern blots. Sequence comparisons argue that ACF7 does not fit into one of the existing families, but represents a new class within the superfamily.

• Lawler J, McHenry K, Duquette M, Derick L
• Characterization of human thrombospondin-4.
• J Biol Chem. 1995; 270: 2809-14
• Display abstract

• The thrombospondins are a family of extracellular calcium binding proteins that are involved in cell proliferation, adhesion, and migration. We have sequenced full-length human thrombospondin-4 and characterized the recombinant protein. In contrast to Xenopus laevis thrombospondin-4, the human protein contains an RGD cell binding sequence in the third type 3 repeat. Transfection of mouse NIH3T3 fibroblasts or C2C12 myoblasts with a full-length human thrombospondin-4 cDNA results in the expression of a polypeptide with a reduced molecular weight of 140,000. In the absence of reducing agent, the expressed protein has an apparent molecular weight of 550,000. Recombinant thrombospondin-4 has been purified from the culture supernatant by heparin-Sepharose and anti-thrombospondin-4 antibody-Affi-gel affinity chromatography. Electron microscopy indicates that thrombospondin-4 is composed of five subunits with globular domains at each end. The observation of a calcium-dependent change in the electron microscopic appearance of thrombospondin-4 is consistent with limited tryptic digestion data that indicate that thrombospondin-4 is resistant to digestion in the presence of calcium. These data indicate that thrombospondin-4 is a pentameric protein that binds to heparin and calcium.

• Winder SJ, Gibson TJ, Kendrick-Jones J
• Dystrophin and utrophin: the missing links!
• FEBS Lett. 1995; 369: 27-33
• Display abstract

• There is considerable sequence homology between dystrophin and utrophin, both at the protein and DNA level, and consequently it was assumed that their domain structures and functions would be similar. As more of the detailed biochemical and cell biological properties of these two proteins become known, so it becomes clear that there are subtle if not significant differences between them. We review recent findings and present new hypotheses into the structural and functional properties of the actin-binding domain, central coiled-coil region and regulatory/membrane protein-binding regions of dystrophin and utrophin.

• Doolittle RF
• The origins and evolution of eukaryotic proteins.
• Philos Trans R Soc Lond B Biol Sci. 1995; 349: 235-40
• Display abstract

• The common ancestry of eukaryotes, archaebacteria and eubacteria is well demonstrated by amino acid sequence comparisons of numerous proteins that are common to all three groups. On the other hand, there are a few proteins, like ubiquitin, that are common to eukaryotes and archaebacteria and which have yet to be observed in eubacteria. Some proteins appear to be wholly restricted to eukaryotes; this is especially true of cytoskeletal proteins. Recently, actin has been found by crystallography to be homologous with an ATP-binding domain found in a heat shock protein and several other proteins common to all three urkingdoms. This observation is puzzling on several counts. Most cytoskeletal proteins like actin and tubulin are very slow changing and must have been so for a very long time. How is it, then, that no sequence resemblance can be discerned with their alledged prokaryotic antecedents? The question is addressed by considering two bacterial fts proteins which appear to be related to actin, on the one hand, and tubulin, on the other. One answer may be that the rate of change of these proteins changed dramatically at a key point in their history. Another possibility is that eukaryotes are much older than some of their other proteins indicate.

• Chuang JZ, Lin DC, Lin S
• Molecular cloning, expression, and mapping of the high affinity actin-capping domain of chicken cardiac tensin.
• J Cell Biol. 1995; 128: 1095-109
• Display abstract

• Tensin, an actin filament capping protein first purified from chicken gizzard, is localized to various types of adherens junctions in muscle and nonmuscle cells. In this paper, we describe the isolation and sequencing of tensin cDNA from a chicken cardiac library. The 6.3-kb chicken cardiac tensin cDNA encodes an open reading frame of 1,792 amino acids. Mammalian cells transfected with the chicken tensin cDNA expressed a polypeptide of approximately 200 kD recognizable by antibodies to chicken gizzard tensin. The expressed protein was incorporated into focal adhesions and other actin-containing structures in the transfected cells. To map the domain associated with tensin's high affinity, barbed-end F-actin-capping activity, bacterially expressed recombinant fusion proteins containing various segments of tensin were prepared and assayed for activity. The results of these experiments show that the high affinity capping domain (kD = 1.3 nM) lies within amino acid residues R1037-V1169. Additional studies on a shorter construct, S1061-H1145, showed that these 85 residues were sufficient for producing complete inhibition of actin polymerization and depolymerization. While this active domain is located within that of the "insertin" sequence (Weigt, C., A. Gaertner, A. Wegner, H. Korte, and H. E. Meyer. 1992. J. Mol. Biol. 227:593-595), our data showing complete inhibition of polymerization and shift in critical concentration are consistent with a simple barbed-end capping mechanism rather than the "insertin model." Our results also differ from those of a recent report (Lo, S. H., P. A. Janmey, J. H. Hartwig, and L. B. Chen. 1994. J. Cell Biol. 125:1067-1075), which concluded that their recombinant tensin has an "insertin-like" inhibitory effect on barbed-end actin polymerization, and that this activity is attributed to residues T936-R1037 (residues 888-989 in their numbering system). In our study, a fusion construct (N790-K1060) encompassing T936-R1037 had no significant effect on actin polymerization and depolymerization, even at high concentrations.

• Edwards RA, Bryan J
• Fascins, a family of actin bundling proteins.
• Cell Motil Cytoskeleton. 1995; 32: 1-9
• Display abstract

• Fascin is an actin-bundling protein that was first isolated from cytoplasmic extracts of sea urchin eggs [Kane, 1975: J. Cell Biol. 66:305-315] and was the first bundling protein to be characterized in vitro. Subsequent work has shown that fascin bundles actin filaments in fertilized egg microvilli and filopodia of phagocytic coelomocytes [Otto et al., 1980: Cell Motil. 1:31-40; Otto and Bryan, 1981: Cell Motil. 1:179-192]. Fifteen years later, the molecular cloning of sea urchin fascin [Bryan et al., 1993: Proc. Natl. Acad. Sci. U.S.A. 90:9115-9119] has led to the identification and characterization of homologous proteins in Drosophila [Cant et al., 1994: J. Cell Biol. 125:369-380], Xenopus [Holthuis et al., 1994: Biochim. Biophys. Acta. 1219:184-188], rodents [Edwards et al,. 1995: J. Biol. Chem. 270:10764-10770], and humans [Duh et al., 1994: DNA Cell Biol. 13:821-827; Mosialos et al., 1994: J. Virol. 68:7320-7328] that bundle actin filaments into structures which stabilize cellular processes ranging from mechanosensory bristles to the filopodia of nerve growth cones. Fascin has emerged from relative obscurity as an exotic invertebrate egg protein to being recognized as a widely expressed protein found in a broad spectrum of tissues and organisms. The purpose of this review is to relate the early studies done on the sea urchin and HeLa cell fascins to the recent molecular biology that defines a family of bundling proteins, and discuss the current state of knowledge regarding fascin structure and function.

• Sheterline P, Clayton J, Sparrow J
• Actin.
• Protein Profile. 1995; 2: 1-103

• Winder SJ, Kendrick-Jones J
• Calcium/calmodulin-dependent regulation of the NH2-terminal F-actin binding domain of utrophin.
• FEBS Lett. 1995; 357: 125-8
• Display abstract

• The cytoskeletal proteins utrophin, dystrophin and alpha-actinin are predicted to form antiparallel dimers thus potentially bringing their NH2-terminal F-actin binding domains in close proximity to their EF-hand containing COOH-terminal domains. This arrangement would allow for calcium-dependent regulation of F-actin binding. We tested this hypothesis by determining the effect of the ubiquitous calcium binding protein calmodulin on their F-actin binding capabilities. Binding of the NH2-terminal F-actin binding domain of utrophin to F-actin was inhibited by increasing concentrations of calmodulin in a calcium-dependent manner. The homologous F-actin binding domains from dystrophin and alpha-actinin were not regulated by calmodulin in the presence or absence of calcium. These findings have implications for the structural organisation of utrophin dimers and also for the replacement of dystrophin by over-expression of utrophin in dystrophic muscle.

• Karpova TS, Tatchell K, Cooper JA
• Actin filaments in yeast are unstable in the absence of capping protein or fimbrin.
• J Cell Biol. 1995; 131: 1483-93
• Display abstract

• Many actin-binding proteins affect filament assembly in vitro and localize with actin in vivo, but how their molecular actions contribute to filament assembly in vivo is not understood well. We report here that capping protein (CP) and fimbrin are both important for actin filament assembly in vivo in Saccharomyces cerevisiae, based on finding decreased actin filament assembly in CP and fimbrin mutants. We have also identified mutations in actin that enhance the CP phenotype and find that those mutants also have decreased actin filament assembly in vivo. In vitro, actin purified from some of these mutants is defective in polymerization or binding fimbrin. These findings support the conclusion that CP acts to stabilize actin filaments in vivo. This conclusion is particularly remarkable because it is the opposite of the conclusion drawn from recent studies in Dictyostelium (Hug, C., P.Y. Jay, I. Reddy, J.G. McNally, P.C. Bridgman, E.L. Elson, and J.A. Cooper. 1995. Cell. 81:591-600). In addition, we find that the unpolymerized pool of actin in yeast is very small relative to that found in higher cells, which suggests that actin filament assembly is less dynamic in yeast than higher cells.

• Schmid MF, Jakana J, Matsudaira P, Chiu W
• Three-dimensional structure of the acrosomal filament of Limulus sperm by 400 kV electron cryomicroscopy.
• Biophys J. 1995; 68: 811-811
• Display abstract

• The acrosomal bundle of Limulus sperm was imaged by electron cryomicroscopy, and the three-dimensional structure of a filament computationally isolated from the bundle was determined by helical image reconstruction. The actin model of Holmes was fit into the map, and its interactions with scruin, the actin-binding and cross-linking protein, were studied. Scruin binds to two consecutive actins along the helix via subdomains 1 and 3. These interactions involve helix-loop-beta motifs that are present in both actin subdomains (in different monomers) in positions available for binding by the same scruin molecule as it wraps around the actin. Taking first the structural motif homology and then looking for sequence pattern similarities, a stretch of 12 out of 20 matches in hydrophobicity is found. Scruin itself has been found to have an internal tandem homology.

• Mezgueldi M, Mendre C, Calas B, Kassab R, Fattoum A
• Characterization of the regulatory domain of gizzard calponin. Interactions of the 145-163 region with F-actin, calcium-binding proteins, and tropomyosin.
• J Biol Chem. 1995; 270: 8867-76
• Display abstract

• Earlier, we proposed that the interaction of gizzard calponin with F-actin, promoting the inhibition of the actomyosin ATPase activity, involves the NH2-terminal portion of the calponin segment Ala145-Tyr182 (Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). In this work, we have directly probed this region for actin binding sites using five peptide analogs covering different stretches of the sequence Thr133-Ile163. Co-sedimentation with F-actin, actomyosin ATPase measurements, and zero-length cross-linking reactions demonstrated that the 19-residue sequence Ala145-Ile163 is essential for actin interaction and ATPase inhibition. Furthermore, each peptide was tested for binding to the Ca(2+)-dependent proteins, caltropin and calmodulin, in both an actomyosin ATPase assay and an affinity chromatographic assay. The results revealed the 11-residue segment Gln153-Ile163, representing the COOH-terminal moiety of the F-actin binding sequence, as a crucial region for the high affinity binding of these regulatory proteins with concomitant removal of the ATPase inhibition. The 153-163 stretch contained also interactive sites for tropomyosin as assessed by affinity chromatography and spectrofluorometry. Collectively, the data support our initial results and highlight the ability of the multifunctional 145-163 region to serve as a potent regulatory domain of the smooth muscle calponin.

• Karpusas M et al.
• 2 A crystal structure of an extracellular fragment of human CD40 ligand.
• Structure. 1995; 3: 1426-1426

• McLaughlin PJ, Weeds AG
• Actin-binding protein complexes at atomic resolution.
• Annu Rev Biophys Biomol Struct. 1995; 24: 643-75
• Display abstract

• This review describes three structures of actin complexed with different monomer-binding proteins, namely with DNase I, gelsolin segment 1, and profilin. In these proteins, the binding sites are discontinuous in the sequence, and those residues that form intermolecular hydrogen bonds are not well conserved in homologous proteins. The strongly conserved residues that define the family of proteins in gelsolin and profilin reflect the underlying structural fold of each. The binding surfaces for segment 1 and profilin are different, although they peripherally overlap on actin. No extreme features in the binding surfaces of these complexes distinguish them from other globular proteins.

• Sasaki T et al.
• Structural characterization of two variants of fibulin-1 that differ in nidogen affinity.
• J Mol Biol. 1995; 245: 241-50
• Display abstract

• Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains 1 (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed KD = 60 nM for variant C and KD > 1 microM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.

• Hori K, Morita F, Matsuzawa F, Aikawa S
• Actin-actin contact: chemical cross-linking between actin and the 2.6-kDa peptide from subdomain 4 of actin.
• J Biochem (Tokyo). 1995; 118: 1232-8
• Display abstract

• Previously, we demonstrated that the 2.6-kDa peptide extending from Arg177 to Tyr198 in subdomain 4 of rabbit skeletal actin bound to actin itself, inhibited the elongation of actin filament, and severed F-actin. The corresponding segment in actin, therefore, is thought to contain the most critical actin-actin contact [Hori, K. and Morita, F. (1992) J. Biochem. 112, 401-408; Hori, K., Itoh, T., Takahashi, K., and Morita, F. (1994) Biochim. Biophys. Acta 1186, 35-42]. In this paper, we report on the binding site in actin for the 2.6-kDa peptide studied by using a zero-length cross-linker, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC). We conducted limited digestion of actin cross-linked with the 125I-labeled 2.6-kDa peptide with various proteases, and developed peptide maps. The cross-linked region of the 2.6-kDa peptide was found to be within the region of Ala114 to Glu167 in actin by identifying the radioactive peptide fragments. The region was further restricted by isolation of radioactive peptide from alpha-chymotryptic digest of the cross-linked actin. The binding site of the 2.6-kDa peptide was finally assigned to be within the 24 amino acid segment from Ala144 to Glu167, which lies in subdomain 3 of actin. Using computer graphics, actin-actin contact provided by the two segments was suggested to be along the left-handed genetic helix of actin filament.

• Kelleher JF, Atkinson SJ, Pollard TD
• Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba.
• J Cell Biol. 1995; 131: 385-97
• Display abstract

• We cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.

• Tsuji FI, Ohmiya Y, Fagan TF, Toh H, Inouye S
• Molecular evolution of the Ca(2+)-binding photoproteins of the Hydrozoa.
• Photochem Photobiol. 1995; 62: 657-61
• Display abstract

• Alignment of the primary structures of the hydrozoan photoproteins, aequorin, mitrocomin, clytin and obelin showed very strong amino acid sequence identities. The Ca(2+)-binding sites of the proteins were found to be highly conserved. The Ca(2+)-binding sites were also homologous to the Ca(2+)-binding sites of other Ca(2+)-binding proteins. However, aequorin, mitrocomin, clytin and obelin differed from other Ca(2+)-binding proteins in that they contained a relatively large number of cysteine, tryptophan, histidine, proline and tyrosine residues, suggesting that these residues may have evolved as part of the light-emitting mechanism. Construction of a phylogenetic tree showed that aequorin, mitrocomin, clytin and obelin form a closely related group of proteins.

• Schnuchel A, Wiltscheck R, Eichinger L, Schleicher M, Holak TA
• Structure of severin domain 2 in solution.
• J Mol Biol. 1995; 247: 21-7
• Display abstract

• The three-dimensional structure of domain 2 of severin in aqueous solution was determined by nuclear magnetic resonance spectroscopy. Severin is a Ca(2+)-activated actin-binding protein that servers F-actin, nucleates actin assembly, and caps the fast-growing ends of actin filaments. The 114-residue domain consists of a central five-stranded beta-sheet, sandwiched between a parallel four-turn alpha-helix and, on the other face, a roughly perpendicular two-turn alpha-helix. There are two distinct binding sites for Ca2+ located near the N and C termini of the long helix. Conserved residues of the gelsolin-severin family contribute to the apolar core of domain 2 of severin, so that the overall fold of the protein is similar to those of segment 1 of gelsolin and profilins. Together with biochemical experiments, this structure helps to explain how severin interacts with actin.

• Pope B, Maciver S, Weeds A
• Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment 4 and identification of calcium binding sites.
• Biochemistry. 1995; 34: 1583-8
• Display abstract

• Gelsolin is composed of six repeating segments of sequence (G1-6) and contains three distinct actin binding sites, two that bind to G-actin and one that binds to filaments. The calcium-dependent actin monomer binding site present in the carboxyl-terminal half of the protein (G4-6) plays a critical role both in the cooperative binding of actin by gelsolin and in its nucleating activity. Here we have localized this actin binding site to segment 4 (G4) by expressing the segments G4, G4-5, G5, and G5-6 in Escherichia coli and analyzing their actin binding properties. In addition we have measured their calcium binding. G4-5 and G5-6 each bind a single calcium ion, but there is no binding by G4 or G5. The affinity of binding by G5-6 is 10 times higher than that of G4-5, and calcium binding by G4-6 shows two sites of different affinity. Thus each actin binding site of gelsolin is restricted to a single segment (G1, G2, and G4), but the nonbinding segments G5 and G6 play an important role in the calcium regulation of actin binding and other activities of gelsolin.

• Amberg DC, Basart E, Botstein D
• Defining protein interactions with yeast actin in vivo.
• Nat Struct Biol. 1995; 2: 28-35
• Display abstract

• Using the two-hybrid protein interaction reporter system, actin, profilin, Srv2p and two SH3-containing proteins are found to bind yeast actin in vivo. When tested for ability to interact with 35 actin mutations distributed over the monomer surface, distinct subsets of mutations characteristic for each putative ligand are found to disrupt binding. In particular, the pattern of differential interactions for the actin-actin interaction is consistent with published structures for the actin filament. Despite functional similarities, the patterns of differential interaction for Srv2p and profilin are different. In contrast, the patterns for profilin and the SH3 domain proteins suggest a shared binding site and commonality in mechanism.

• Pignol D, Bertrand JA, Bernard JP, Verdier JM, Dagorn JC, Fontecilla-Camps JC
• Crystallization and preliminary crystallographic study of human lithostathine.
• Proteins. 1995; 23: 604-6
• Display abstract

• Crystals of human lithostathine, a pancreatic glycoprotein which inhibits the growth and nucleation of calcium carbonate crystals, were grown using PEG 4000 as the precipitating agent. The crystals belong to the hexagonal space group P6(1) (or its enantiomorph P6(5)) and diffract to 1.55 A resolution. There is one molecule in the asymmetric unit and the crystals have 39% solvent.

• Discher DE, Winardi R, Schischmanoff PO, Parra M, Conboy JG, Mohandas N
• Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening.
• J Cell Biol. 1995; 130: 897-907
• Display abstract

• Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function.

• Way M, Sanders M, Garcia C, Sakai J, Matsudaira P
• Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.
• J Cell Biol. 1995; 128: 51-60
• Display abstract

• The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

• Bonet-Kerrache A, Mornet D
• Importance of the C-terminal part of actin in interactions with calponin.
• Biochem Biophys Res Commun. 1995; 206: 127-32
• Display abstract

• Native, modified or trypsin-truncated actin was used to study the impact of modifying the C-terminal part of actin (the last three amino acids) on interactions with calponin. We used three different techniques to show that these amino acids are essential for the actin-calponin interface. The locations of actin-calponin interaction sites on the actin crystal are discussed in terms of previously reported data.

• Stafford WF 3rd, Mabuchi K, Takahashi K, Tao T
• Physical characterization of calponin. A circular dichroism, analytical ultracentrifuge, and electron microscopy study.
• J Biol Chem. 1995; 270: 10576-9
• Display abstract

• Calponin is a thin filament-associated smooth muscle protein that has been suggested to play a role in the regulation of smooth muscle contraction. We have used circular dichroism spectroscopy, electron microscopy, and analytical ultracentrifugation to study the physical properties of recombinant chicken gizzard alpha-calponin. The alpha-helix content of alpha-calponin was estimated from its circular dichroism spectrum to be approximately 13%, alpha-Calponin melts with a single sharp transition at approximately 57 degrees C. Rotary shadowing electron micrographs of alpha-calponin reveal diverse shapes ranging from elongated rods to collapsed coils. The lengths of the rod-shaped structures are approximately 18 nm. Analytical ultracentrifugation studies found alpha-calponin to be homogeneous with a monomer molecular mass of 31.4 kDa, and a s20,w value of 2.34 S. These data could be used to model alpha-calponin as a prolate ellipsoid of revolution with an axial ratio of 6.16, a length of 16.2 nm, and a diameter of 2.6 nm. Taken together, our results indicate that calponin is a flexible, elongated molecule whose contour length is sufficient to span three actin subunits along the long pitch helix of an F-actin filament.

• Rashid DJ, Wedaman KP, Scholey JM
• Heterodimerization of the two motor subunits of the heterotrimeric kinesin, KRP85/95.
• J Mol Biol. 1995; 252: 157-62
• Display abstract

• The heterotrimeric kinesin-related motor protein, KRP85/95 is assembled from two kinesin-related polypeptides, SpKRP85 and SpKRP95, together with an uncharacterized 115 kDa polypeptide. Here we report the deduced amino acid sequence of SpKRP95, a close relative of SpKRP85. Both SpKRP85 and SpKRP95 are predicted to have a tripartite domain organization consisting of an N-terminal motor domain, a central stalk domain capable of coiled-coil formation, and a second globular C-terminal domain. The sequences of the central stalk domains predict that SpKRP85 and SpKRP95 should be capable of forming heterodimeric coiled coils. Furthermore, SpKRP85-SpKRP95 complexes can be immunoprecipitated from a cell-free translation system, providing direct evidence that SpKRP85 and SpKRP95 are capable of heterodimerization.

• Petrova TV, Comte M, Takagi T, Cox JA
• Thermodynamic and molecular properties of the interaction between amphioxus calcium vector protein and its 26 kDa target.
• Biochemistry. 1995; 34: 312-8
• Display abstract

• Calcium vector protein (CaVP) of amphioxus shares some common structural features with Ca(2+)-dependent activators such as troponin C and calmodulin, and is associated in vivo with a 26 kDa (CaVPT), a multidomain protein with one IQ- and two IgII-motifs. Isolated CaVP binds two Ca2+ ions with very different intrinsic affinity constants: K'Ca1 = 4.9 x 10(6) M-1 and K'Ca2 = 7.3 x 10(3) M-1, respectively. In the complex with CaVPT, CaVP also binds two Ca2+, but with strong positive cooperativity (nH = 1.9) and with distinctly higher affinity: K'Ca1 = 2.4 x 10(5) M-1 and K'Ca2 = 1.0 x 10(8) M-1. Since neither in the isolated CaVP nor in the complex Ca2+ binding is influenced by 2 mM MgCl2, both sites can be considered as Ca(2+)-specific. In the absence of Ca2+, the complex is stable under physiological conditions, but the interaction is governed by the principle of linked functions and Ca2+ binding to CaVP reinforces the affinity between CaVP and CaVPT 70-fold. Both proteins interact with the hydrophobic probe 2 p-toluidinylnaphthalene-6-sulfonate (TNS), but CaVPT enhances the fluorescence 45-fold, CaVP-Ca2 and metal-free CaVP only 10- and 5-fold, respectively. Complex formation between CaVPT and CaVP leads to a 3-fold reduction of the fluorescence enhancement, suggesting that a strong solvent-shielded hydrophobic core is formed. CaVP contains two highly reactional thiols (kSH > 0.3 s-1) for 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB); CaVPT contains three thiols, two of them also with kSH > 0.3 s-1 in the native state.(ABSTRACT TRUNCATED AT 250 WORDS)

• Egelman EH
• Actin filament structure. The ghost of ribbons past.
• Curr Biol. 1994; 4: 79-81
• Display abstract

• The high-resolution structures now available for monomeric actin, and for the actin-binding proteins profilin, gelsolin and myosin, have important implications for structural models of the actin filament.

• Hori K, Morita F
• [Molecular mechanism of actin-actin contact]
• Tanpakushitsu Kakusan Koso. 1994; 39: 871-6

• Markus MA, Nakayama T, Matsudaira P, Wagner G
• 1H, 15N, 13C and 13CO resonance assignments and secondary structure of villin 14T, a domain conserved among actin-severing proteins.
• J Biomol NMR. 1994; 4: 553-74
• Display abstract

• Sequence-specific assignments have been made for the 1H, 15N, 13C and 13CO resonances of 14T, the 126-residue amino-terminal domain of the actin-severing protein villin. Villin is a member of a family of proteins that regulate cytoskeletal actin by severing, capping and nucleating actin filaments. Actin binding is dependent on calcium and disrupted by phosphatidyl inositol 4,5-bisphosphate. Actin-severing proteins are built from three or six repeats of a conserved domain, represented by 14T. Expression in Escherichia coli facilitated incorporation of 15N and 13C isotopes and application of triple-resonance, backbone-directed strategies for the sequential assignments. Elements of regular secondary structure have been identified by characteristic patterns of NOE cross peaks and values of vicinal 3JHNH alpha coupling constants. Amide protons that exchange slowly (rates less than 1.0 x 10(-4) per min) are concentrated in the central beta-sheet and the second and third alpha-helices, suggesting that these elements of secondary structure form very stable hydrogen bonds. Assignments for the amide nitrogens and protons have been examined as a function of pH and calcium concentration. Based on the conservation of chemical shifts in the core of the domain, villin 14T maintains the same overall fold in the pH range from 4.15 to 6.91 and the calcium range from 0 to 50 mM. The calcium data indicate the presence of two calcium-binding sites and suggest their locations.

• David C, Vigues B
• Calmyonemin: a 23 kDa analogue of algal centrin occurring in contractile myonemes of Eudiplodinium maggii (ciliate).
• Cell Motil Cytoskeleton. 1994; 27: 169-79
• Display abstract

• Myonemes are bundles of thin filaments (3-6 nm in diameter) which mediate calcium-induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calcium-binding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium-modulated contractile protein centrin. Western-blot analysis indicates that the 23 kDa protein cross-reacts antigenically with anti-centrin antibodies. In addition, the 23 kDa protein displays calcium-induced changes in both electrophoretic and chromatographic behaviour, and contains calcium-binding domains that conform to the EF-hand structure, as known for centrin. Based on these observations, we conclude that a calcium-binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myoneme-mediated retraction of the ciliature.

• Kobayashi R, Kubota T, Hidaka H
• Purification, characterization, and partial sequence analysis of a new 25-kDa actin-binding protein from bovine aorta: a SM22 homolog.
• Biochem Biophys Res Commun. 1994; 198: 1275-80
• Display abstract

• We have purified a Ca(2+)-sensitive 25-kDa protein from bovine aorta. The 25-kDa protein remains associated with the membrane fraction in the presence of Ca2+ and is dissociated by EGTA. The purified protein binds directly to F-actin at a ratio of 1:6 actin monomers, with a binding constant of 7.0 x 10(5) M-1. The partial sequence analysis revealed a high homology to predicted protein derived from mRNA, named WS3-10 and chicken SM22 protein. Although chicken SM22 had not any interaction with contractile proteins or Ca2+, the bovine homolog clearly binds to F-actin and has Ca(2+)-binding domain in its primary structure.

• Van Der Bent A, Ijzerman AP, Soudijn W
• Molecular modelling of CCK-A receptors.
• Drug Des Discov. 1994; 12: 129-48
• Display abstract

• Recently, the primary structure of the cholecystokinin A-type (CCK-A) receptor has been determined. From the Kyte-Doolittle-predicted hydrophobic stretches of this sequence and the transmembrane domains of bacteriorhodopsin, a membrane-bound protein of known tertiary structure, a three-dimensional model of the membrane-embedded part of this receptor was built. Subsequently, the modelled receptor pore was searched for a binding site that matches the structural and conformational characteristics of the parent classes of the antagonists devazepide and lorglumide. In addition, the binding mode of hybrid analogues of these reference compounds was examined. The proposed antagonist, binding site includes regions in which hydrophobic, hydrogen-bonding and aromatic interactions stabilize the receptor-ligand complex.

• Howard T, Li Y, Torres M, Guerrero A, Coates T
• The 47-kD protein increased in neutrophil actin dysfunction with 47- and 89-kD protein abnormalities is lymphocyte-specific protein.
• Blood. 1994; 83: 231-41
• Display abstract

• A male child born of related parents suffered recurrent infections because of neutrophil actin dysfunction with increased amounts of a 47-kD protein and decreased amounts of an 89-kD protein (NAD 47/89). The patient and family members were studied to define the nature of the abnormal proteins and to examine their role in the functional defects of neutrophil actin dysfunction (NAD) 47/89 polymorphonuclear neutrophils (PMNs). NAD 47/89 PMNs are defective in motility, microfilamentous cytoskeletal structure, and formyl peptide-induced actin polymerization and express increased amounts of a 47-kD protein and decreased amounts of an 89-kD proteins intermediate abnormality in amount of 47-kD and 89-kD proteins in PMNs from parents and a female sibling suggest the disease is an autosomal recessive disorder. Immunoblots with monoclonal antibody (MoAb1) and polyclonal antibody raised to 47-kD protein showed the 89-kD protein is antigenically distinct from the 47-kD protein and the 89-kD protein is not gelsolin. 125I-actin binding to one-dimensional (1 D) and 2 D gels of PMN proteins from NAD 47/89 proband, family members, and controls showed the 47-kD protein binds actin, is acidic (pl = 4.5 to 4.7), is recognized by the MoAb1, exists on 2-D gels as three distinct actin binding species (MWapp 52 kD, 47-kD, and 44-kD), and is present in control PMNs in lesser amount than in PMNs of NAD 47/89 proband or parents. Immunoaffinity purification of the 47 kD actin binding protein on MoAb1 matrix yielded a multimolecular complex with proteins of MWapp 180 kD, 71 kD, 47 kD and actin. Cloning, sequencing, and expression of a 1.58-kb cDNA selected for MoAb1 reactivity from a HL60 expression library and microsequence of native PMNs, 47-kD actin binding protein showed the overexpressed 47-kD protein is lymphocyte-specific protein 1 (LSP1), which is a known actin binding protein. The results show LSP1 is expressed in PMNs and suggest overexpression of LSP1 is related to the motility and cytoskeletal abnormalities in NAD 47/89 PMNs.

• Young CL, Feierstein A, Southwick FS
• Calcium regulation of actin filament capping and monomer binding by macrophage capping protein.
• J Biol Chem. 1994; 269: 13997-4002
• Display abstract

• Macrophage capping protein (MCP) is a unique member of the gelsolin-villin family of calcium-activated barbed end capping proteins which in micromolar Ca2+ also binds actin monomers and nucleates actin assembly. Unlike gelsolin, MCP cannot sever actin filaments, and its Ca(2+)-dependent interaction with actin is completely reversible. The Ca2+ binding properties of MCP and its Ca(2+)-dependent functions were studied quantitatively. MCP undergoes a Ca(2+)-induced conformational change as evidenced by different chymotryptic digest patterns in 0.2 mM CaCl2 compared with 2 mM EGTA. MCP has a single low affinity Ca2+ binding site (KD = 37 microM). Binding of MCP to actin monomers requires a similar Ca2+ concentration ([Ca2+]0.5 = 62 microM) suggesting that MCP.Ca2+ complex formation promotes monomer binding. In contrast, filament capping by MCP requires 1/60th of the Ca2+ concentration required for monomer binding, half-maximal capping occurring at 1 microM Ca2+. The marked difference in the Ca2+ sensitivity of these two functions indicate that MCP's primary actin regulatory role in the macrophage is likely to be capping of the barbed ends of actin filaments.

• Pope B, Way M, Matsudaira PT, Weeds A
• Characterisation of the F-actin binding domains of villin: classification of F-actin binding proteins into two groups according to their binding sites on actin.
• FEBS Lett. 1994; 338: 58-62
• Display abstract

• The F-actin binding properties of chicken villin, its headpiece and domains 2-3 (V2-3) have been analysed to identify sites involved in bundle formation. Headpiece and V2-3 bind actin with Kd values of approximately 7 microM and approximately 0.3 microM, respectively, at low ionic strength. V2-3 binding, like that of villin, is weakened with increasing salt concentration; headpiece binding is not. Competition experiments show that headpiece and V2-3 bind to different sites on actin, forming the two cross-linking sites of villin. Headpiece does not compete with the F-actin binding domains of gelsolin or alpha-actinin, but it dissociates actin depolymerizing factor. We suggest that the F-actin binding domains of actin severing, crosslinking and capping proteins can be organized into two classes.

• Schmid MF, Agris JM, Jakana J, Matsudaira P, Chiu W
• Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin.
• J Cell Biol. 1994; 124: 341-50
• Display abstract

• Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits.

• Schutt CE, Rozycki MD, Lindberg U
• What's the matter with the ribbon?
• Curr Biol. 1994; 4: 185-6

• Hitt AL, Lu TH, Luna EJ
• Ponticulin is an atypical membrane protein.
• J Cell Biol. 1994; 126: 1421-31
• Display abstract

• We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

• Safer D, Nachmias VT
• Beta thymosins as actin binding peptides.
• Bioessays. 1994; 16: 590-590

• Matsudaira P
• The fimbrin and alpha-actinin footprint on actin.
• J Cell Biol. 1994; 126: 285-7

• Holtzman DA, Wertman KF, Drubin DG
• Mapping actin surfaces required for functional interactions in vivo.
• J Cell Biol. 1994; 126: 423-32
• Display abstract

• An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin.

• Civelekoglu G, Edelstein-Keshet L
• Modelling the dynamics of F-actin in the cell.
• Bull Math Biol. 1994; 56: 587-616
• Display abstract

• The regulation of the interactions between the actin binding proteins and the actin filaments are known to affect the cytoskeletal structure of F-actin. We develop a model depicting the formation of actin cytoskeleton, bundles and orthogonal networks, via activation or inactivation of different types of actin binding proteins. It is found that as the actin filament density increases in the cell, a spontaneous tendency to organize into bundles or networks occurs depending on the active actin binding protein concentration. Also, a minute change in the relative binding affinity of the actin binding proteins in the cell may lead to a major change in the actin cytoskeleton. Both the linear stability analysis and the numerical results indicate that the structures formed are highly sensitive to changes in the parameters, in particular to changes in the parameter phi, denoting the relative binding affinity and concentration of the actin binding proteins.

• Otto JJ
• Actin-bundling proteins.
• Curr Opin Cell Biol. 1994; 6: 105-9
• Display abstract

• Recent studies have greatly expanded our understanding of actin-bundling proteins. A new group of actin-bundling proteins, the fascins, has been recognized. An actin-bundling protein inhibits actin depolymerization even under conditions in which it cannot produce a gel, which suggests that bundling proteins may affect actin filament dynamics. A villin-like protein is present in Dictyostelium, shedding doubt on current ideas on the evolution of villin. Domain mapping continues to be a major thrust of research into most groups of bundling proteins.

• Rozycki MD, Myslik JC, Schutt CE, Lindberg U
• Structural aspects of actin-binding proteins.
• Curr Opin Cell Biol. 1994; 6: 87-95
• Display abstract

• The three-dimensional structures of myosin subfragment 1 (S1), gelsolin segment 1 complexed with alpha-actin, villin fragment 14T, Acanthamoeba profilin-I, and bovine profilin complexed with beta-actin were completed last year. Together, they expand our understanding of the structural organization of actin-binding proteins. In addition, the segment 1 and bovine profilin complexes provide atomic-level descriptions of their interfaces with actin.

• Corrado K, Mills PL, Chamberlain JS
• Deletion analysis of the dystrophin-actin binding domain.
• FEBS Lett. 1994; 344: 255-60
• Display abstract

• Three sequence motifs at the N-terminus of dystrophin have previously been proposed to be important for binding to actin. By analyzing a series of purified bacterial fusion proteins deleted for each of these sites we have demonstrated that none of the three are critical for dystrophin-actin interactions. Instead, our data suggest that sequences in the N-terminal 90 amino acids of dystrophin, excluding a conserved KTFT motif, contain the major site for interaction with actin.

• Procyshyn RM, Reid RE
• An examination of glutamic acid in the -X chelating position of the helix-loop-helix calcium binding motif.
• Arch Biochem Biophys. 1994; 311: 425-9
• Display abstract

• Poor calcium affinity was exhibited in helix-loop-helix calcium binding motifs with X-axis acid pairs containing aspartic acid in the -X chelating position. In order to increase interaction of the -X chelating residue with the cation, helix-loop-helix calcium binding motifs were synthesized containing three and four acid residues in chelating positions, with a glutamic acid replacing aspartic acid in the -X chelating position. The glutamate-containing motif gave an unexpected 6-fold decrease in cation affinity for the three-acid residue loop motif (KCa = 524 microM vs KCa = 3140 microM) and a 46-fold decrease for the four-acid residue loop motif (KCa = 42.1 microM vs KCa 1950 microM). To improve calcium binding of the glutamate-containing motifs, peptides were synthesized keeping glutamate in the -X position and inserting serine in the +Z position to provide a hydrogen-bonded system stabilizing the glutamate interaction with the cation. The serine residue further reduced calcium affinity in both the three-acid residue loop (KCa = 19.6 mM) and the four-acid residue loop (KCa = 2806 microM). These results indicate that glutamate and serine residues in the -X and +Z positions, respectively, can be detrimental to calcium binding. However, in natural calcium binding proteins, glutamate in the -X chelating position can confer high affinity for calcium in helix-loop-helix calcium binding motifs, but this may be dependent on the environment created by as yet undetermined factors.

• Baumann U, Wu S, Flaherty KM, McKay DB
• Three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa: a two-domain protein with a calcium binding parallel beta roll motif.
• EMBO J. 1993; 12: 3357-64
• Display abstract

• The three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa, a zinc metalloprotease, has been solved to a resolution of 1.64 A by multiple isomorphous replacement and non-crystallographic symmetry averaging between different crystal forms. The molecule is elongated with overall dimensions of 90 x 35 x 25 A; it has two distinct structural domains. The N-terminal domain is the proteolytic domain; it has an overall tertiary fold and active site zinc ligation similar to that of astacin, a metalloprotease isolated from a European freshwater crayfish. The C-terminal domain consists of a 21-strand beta sandwich. Within this domain is a novel 'parallel beta roll' structure in which successive beta strands are wound in a right-handed spiral, and in which Ca2+ ions are bound within the turns between strands by a repeated GGXGXD sequence motif, a motif that is found in a diverse group of proteins secreted by Gram-negative bacteria.

• Vinayagamoorthy T, Dakour J, Dixon W, Jimbow K
• cDNA-based functional domains of a calnexin-like melanosomal protein, p90.
• Melanoma Res. 1993; 3: 263-9
• Display abstract

• We have recently identified a gene encoding a calnexin-like protein (p90) by the immunoscreening of a human melanoma cDNA library, using a rabbit anti-human melanosomal antibody. This p90 protein was highly expressed by human melanocytes and associated with melanosomal membrane and endoplasmic reticulum. In this study we report the computer analysis of the predicted amino acid sequence of this calnexin-like melanosomal protein. We found that p90 is a membrane-bound protein whose large N-terminal domain is located within the melanosomal compartment; its shorter C-terminal is exposed to the cytosol and separated by a short transmembrane region. This p90 protein was found to have consensus sequences of a Ca(2+)-binding loop and a protein kinase C phosphorylation site at the N-terminal domain. The C-terminal domain, on the other hand, contained sequences of a casein kinase II phosphorylation site and two protein kinase A phosphorylation sites. Such functional motifs could provide signal transduction across the melanosomal membrane, the reception of melanogenic protein via carriers at the melanosomal membrane and the translocation of melanosomes in the melanocyte.

• Hessian PA, Edgeworth J, Hogg N
• MRP-8 and MRP-14, two abundant Ca(2+)-binding proteins of neutrophils and monocytes.
• J Leukoc Biol. 1993; 53: 197-204
• Display abstract

• Two calcium-binding proteins, named migration inhibitory factor-related proteins-8 (MRP-8) and MRP-14, are primarily expressed by circulating human neutrophils and monocytes. Evidence accumulating from the investigations of several independent groups is now leading to an improved understanding of the biology of these proteins. Both MRP-8 and MRP-14 display features characteristic of members of the S100 family of calcium-binding proteins. Some of these features predict functions for MRP-8 and MRP-14 but to date an exact and well-defined function remains elusive. Here we review the available information and highlight evidence that suggests the function of MRP-8 and MRP-14 may be associated with both monocyte and neutrophil activation and the accumulation of these cells in inflammatory sites.

• Minor W, Steczko J, Bolin JT, Otwinowski Z, Axelrod B
• Crystallographic determination of the active site iron and its ligands in soybean lipoxygenase L-1.
• Biochemistry. 1993; 32: 6320-3
• Display abstract

• Five ligands of the active site iron atom in soybean lipoxygenase L-1 have been identified from the electron density map of the crystallized enzyme. The position of the iron atom can be readily and independently located from an anomalous difference electron density map. The ligands identified are His-499, His-504, His-690, Asn-694, and Ile-839, the carboxy-terminal residue. Our previous view that these three histidines are essential for activity and binding of iron, based on site-specific mutation studies, is confirmed. A sixth protein ligand is not present, and the sixth coordination site opens into a wide cleft. The structure of the soybean lipoxygenase was solved by multiple anomalous isomorphous replacements.

• Hofmann A, Noegel AA, Bomblies L, Lottspeich F, Schleicher M
• The 100 kDa F-actin capping protein of Dictyostelium amoebae is a villin prototype ('protovillin').
• FEBS Lett. 1993; 328: 71-6
• Display abstract

• The 100 kDa actin-binding protein from Dictyostelium amoebae is an F-actin capping protein that displays neither severing nor crosslinking nor nucleating activities [Hofmann et al. (1992) Cell Motil. Cytoskel. 23,133-144]. Cloning and sequencing of the gene revealed that the protein is highly homologous to vertebrate villin, a unique component of brush border microvilli and contains six domains fused to a villin-like headpiece domain via a threonine/proline rich neck region. The functional differences and similarities between the 100 kDa protein and villin are reflected in the amino acid sequences. We draw from the data the following conclusions. (i) The presence of a six domain protein in Dictyostelium suggests that in contrast to the current view gene duplications must have happened before Dictyostelium branched off during evolution. (ii) The villin-like molecule in Dictyostelium appears to be a premature villin ('protovillin') which is able to cap actin filaments but still lacks the other villin-type actin-binding activities. This renders capping of actin filaments as the evolutionarily oldest function of an F-actin binding protein.

• Bewley MC, Boustead CM, Walker JH, Waller DA, Huber R
• Structure of chicken annexin V at 2.25-A resolution.
• Biochemistry. 1993; 32: 3923-9
• Display abstract

• The crystal structure of chicken annexin V has been solved by molecular replacement and refined at 2.25 A. The final R factor is 19.7% with good geometry. The chicken annexin V structure is very similar to the human annexin V structure, with four similar domains each containing five helices. The structure includes three calcium ions in domains I, II, and IV, each bound by the characteristic K-G-X-G-T-(38 residues)-D/E motif. In view of the structural similarity between human and chicken annexin V, we suggest that they have a common vital function which developed early in evolutionary history.

• Messier JM, Shaw LM, Chafel M, Matsudaira P, Mercurio AM
• Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages.
• Cell Motil Cytoskeleton. 1993; 25: 223-33
• Display abstract

• The actin-bundling protein fimbrin is homologous to 1-plastin, a 65kD phosphoprotein expressed in leukocytes and transformed cells [de Arruda et al., J. Cell Biol. 111, 1069-1080]. Because fimbrin is present in cell adhesion sites, we studied the phosphorylation state of fimbrin and its distribution in macrophages sequentially extracted with Triton-X-100 (soluble fraction), Tween 40-deoxy-cholate (cytoskeletal fraction), and SDS (insoluble cytoskeletal fraction). The approximate distribution of fimbrin and actin among these fractions was found to be: 65% fimbrin/55% actin in the soluble fraction, 30% fimbrin/20% actin in the cytoskeletal fraction, and 5% fimbrin/25% actin in the insoluble cytoskeletal fraction. PMA did not alter this distribution. Fluorescence microscopy of acetone-extracted macrophages showed that actin is concentrated in podosomes at the substratum interface and is diffusely distributed throughout the remainder of the cell. Fimbrin colocalizes with actin in podosomes and also exhibits a punctate distribution in the cytoplasm that overlaps with actin. In Tween 40/DOC-extracted cells, podosomes remain, and fimbrin also exhibits a punctate distribution along actin filaments. Metabolic 32PO4 labeling revealed that fimbrin is constitutively phosphorylated and that phosphorylated fimbrin is concentrated in the insoluble cytoskeletal fraction. PMA increased the relative levels of fimbrin phosphorylation twofold but did not alter the pattern of fimbrin fluorescence or the distribution of phosphorylated fimbrin. Limited trypsin digestion and phosphoamino acid analysis demonstrated that phosphorylation occurs specifically on serine residues within the 10kD headpiece domain of fimbrin. Phosphorylation of the headpiece domain could regulate the actin binding and bundling properties of fimbrin, or it could regulate the interaction of fimbrin with other proteins.

• Koepf EK, Burtnick LD
• Horse plasma gelsolin labelled with fluorescein isothiocyanate responds to calcium and actin.
• Eur J Biochem. 1993; 212: 713-8
• Display abstract

• Reaction between horse plasma gelsolin and fluorescein-5-isothiocyanate (FITC) resulted in incorporation of 4.8 +/- 0.6 fluorescein groups/gelsolin molecule. The sites of modification were not clustered in any one portion of the gelsolin polypeptide chain; all major peptides produced by proteolytic digestion with alpha-chymotrypsin exhibited a fluorescence characteristic of fluorescein. FITC-gelsolin has a peptide-backbone circular dichroism spectrum at 20 degrees C that is indistinguishable from that of native gelsolin, but FITC-gelsolin is considerably more resistant than native gelsolin to thermally induced precipitation. FITC-gelsolin is fully able to carry out severing of F-actin filaments, the prime function of gelsolin in plasma. An opening up of the structure of gelsolin on binding Ca2+ is evident from an increased susceptibility of FITC-gelsolin to quenching by I-. Ca2+ dependence of the interaction between gelsolin and actin is evident in titrations both of intensity and polarization of the fluorescence of FITC-gelsolin solutions. A Ca(2+)-sensitive interaction between gelsolin and tropomyosin also is observed.

• Nachmias VT
• Small actin-binding proteins: the beta-thymosin family.
• Curr Opin Cell Biol. 1993; 5: 56-62
• Display abstract

• Thymosin beta 4 is a major actin monomer binding protein present at high concentration in many vertebrate cells and cell lines. The interactions of actin with thymosin beta 4, actobindin and profilin are compared. Nine beta-thymosins have been identified; six have been shown to bind to actin. Regulation of the synthesis of thymosin beta 10 and thymosin beta 4 has been found in brain development and after stimulation of several cell types, respectively. The extracellular effects of thymosin beta 4 still need clarification.

• Censullo R, Cheung HC
• A rotational offset model for two-stranded F-actin.
• J Struct Biol. 1993; 110: 75-83
• Display abstract

• We propose the following "rotational offset" model for two independent strands of F-actin to account for the observation that it is possible, at times, for the crossover repeat to either alternate between long and short periods or remain constant (Bremer et al., 1991. J. Cell Biol. 115, 689-703). Rotational offset is the manifestation of the angular component of "lateral slipping" between the two long-pitch, right-handed strands comprising the actin filament. The present model is based on the premise that the longitudinal bond connecting the subunits along a single long-pitch strand is substantially stronger than the diagonal bond that connects interstrand subunits. We pose that, over fairly long stretches, the backbones of the two right-handed strands are individually close to being ideal helices, and that it is possible for the backbone of one strand to "roll across" the other. The rotational offset angle (epsilon 0) is the amount that one helical strand is angularly displaced relative to the position that otherwise would allow the two strands to be described as an ideal single genetic helix. Such an independent movement of the two strands is shown to shift the monomers that are involved in crossover points and produce the different patterns in crossover periods which have been observed from electron micrographs analysis. We specifically demonstrate that for a constant nonzero rotational offset the length of the crossover periods alternates, whereas for a constant offset of zero the crossover period remains constant. We also show that changes in the rotational offset angle along the filament can account for variable (random) crossover periods.

• Lamb JA, Allen PG, Tuan BY, Janmey PA
• Modulation of gelsolin function. Activation at low pH overrides Ca2+ requirement.
• J Biol Chem. 1993; 268: 8999-9004
• Display abstract

• The activation of gelsolin by calcium has been postulated to be involved in the receptor-mediated reorganization of the actin cytoskeleton, but cytoskeletal reorganization can also occur in cells with intracellular Ca2+ clamped at nanomolar levels. Fluorescence measurements using Fura-2 show that at pH 7.4, the Ca2+ requirement for gelsolin activation in vitro is higher than previously reported, with half-maximal activation of severing and nucleation occurring at 10 microM Ca2+. The Ca2+ requirement for gelsolin activity decreases at more acid pH and is approximately 3 microM at pH 6.5. At pH below 6.0, gelsolin no longer requires Ca2+ for activity and severs actin filaments, binds two actin monomers, and nucleates filament formation in EGTA-containing solutions. The pH-activated severing activity is inhibited by mixed lipid vesicles containing phosphatidylinositol 4,5-bisphosphate. A Ca(2+)-sensitive fragment consisting of the first 135 amino acids of human cytoplasmic gelsolin also demonstrates severing activity at pH < 6.0 in the absence of Ca2+. In contrast, the gelsolin homologs severin and villin maintain Ca2+ regulation of severing activity at low pH. These differences suggest that activation of gelsolin at low pH cannot be explained merely by destabilization of F-actin. The difference in diffusion constants of gelsolin measured at pH 5.5 and 6.5, as determined by dynamic light scattering, suggests that the molecule undergoes a shape change similar to that reported upon binding Ca2+ at neutral pH. These results suggest a mechanism by which gelsolin may be activated in vivo under conditions where Ca2+ transients do not occur.

• Janmey P
• Cell biology. A slice of the actin.
• Nature. 1993; 364: 675-6

• Bernard JP, Takacs T, de Reggi M, Sarles H, Dagorn JC
• [Pancreatic lithostathine inhibitor of calcium carbonate precipitation: structure-function relationship]
• Nephrologie. 1993; 14: 257-9
• Display abstract

• Pancreatic juice is naturally supersatured in calcium and bicarbonate ions. A mechanism controlling CaCO3 crystal formation and growth is therefore necessary to prevent duct clogging. Lithostathine, a glycoprotein synthesized by acinar cells and secreted in pancreatic juice, could be involved in such a control. Lithostathine significantly delayed crystal nucleation and inhibited growth of CaCO3 crystals from supersatured solutions. Lithostathine adsorbed to sites specifically inhibiting crystal growth with a dissociation constant Kd = 0.9 x 10(-6) mol/L. The glycosylated N-terminal undecapeptide generated by limited trypsin hydrolysis of lithostathine, inhibited CaCO3 crystal growth with a Kd = 3.4 x 10(-6) mol/L similar to that of lithostathine. On the contrary, the carboxy-terminal polypeptide (lithostathine H) was inactive. The N-terminal undecapeptide of lithostathine is therefore essential to the inhibitory activity of the protein on CaCO3 crystal growth.

• Dufort PA, Lumsden CJ
• Cellular automaton model of the actin cytoskeleton.
• Cell Motil Cytoskeleton. 1993; 25: 87-104
• Display abstract

• We describe a cellular automaton model of the actin cytoskeleton. The model incorporates spatial and temporal behavior at the macromolecular level and is relevant to the viscous nonequilibrium conditions suspected to occur in vivo. The model includes cation and nucleotide binding to actin monomers, actin nucleation and polymerization into filaments, cross-linking with alpha-actinin, monomer sequestration with profilin, filament severing, capping and nucleation with gelsolin, binding of profilin and gelsolin to membrane-bound phosphatidylinositide biphosphate (PIP2), and regulation of cross-linking and severing by changing calcium levels. We derive 1) equations for the molecular translation and rotation probabilities required for the cellular automaton simulation in terms of molecular size, shape, cytoplasmic viscosity, and temperature; and 2) equations for the binding probabilities of adjacent molecules in terms of experimentally determined reaction rate constants. The model accurately captures the known characteristics of actin polymerization and subsequent ATP hydrolysis under different cation and nucleotide conditions. An examination of gelation and sol-gel transitions resulting from calcium regulation of alpha-actinin and gelsolin predicts an inhomogeneous distribution of bound alpha-actinin and F-actin. The double-bound alpha-actinin (both ends bound to F-actin) is tightly bunched, while single-bound alpha-actinin is moderately bunched and unbound alpha-actinin is homogeneously distributed. The spatial organization of the alpha-actinin is quantified using estimates of fractal dimension. The simulation results also suggest that actin/alpha-actinin gels may shift from an isotropic to an amorphous phase after shortening of filaments. The gel-sol transition of the model shows excellent agreement with the present theory of polymer gels. The close correspondence of the model's predictions with previous experimental and theoretical results suggests that the model may be pertinent to better understanding the spatial and temporal properties of complex cytoskeletal processes.

• Pacaud M, Derancourt J
• Purification and further characterization of macrophage 70-kDa protein, a calcium-regulated, actin-binding protein identical to L-plastin.
• Biochemistry. 1993; 32: 3448-55
• Display abstract

• We have previously identified a macrophage 70-kDa, actin-bundling protein as a constituent of actin-based cytoplasmic gel and showed that its association with or dissociation from cytoplasmic gels was remarkably affected by submicromolar calcium. In this study, we purified the 70-kDa protein from soluble cytosolic extracts and carried out a more detailed characterization. The amino acid sequences of four peptidic fragments, obtained from the purified protein by enzymatic or chemical cleavage, were completely or nearly identical to those of L-plastin, a protein initially identified in transformed cells from solid tumors (Goldstein & Leavitt, 1985). By Western blot analysis of normal cells and tissues using specific anti-70-kDa protein antibodies, the 70-kDa molecule was detected only in hematopoietic cells. The 70-kDa protein bound to actin with apparent Kd values of 1.8 and 5.5 microM in the absence and presence of 20 microM free calcium, respectively. Cross-linking activity measured by falling-ball viscosimetry was optimal at free calcium lower than 0.15 microM but was progressively inhibited at higher calcium concentrations, within the physiological range. Half-maximal inhibition occurred at 1.6 microM free calcium. No severing of actin filaments by the 70-kDa protein was observed in any of these assays or previously (Pacaud & Harricane, 1987). Major conformational changes of the protein, as measured by the fluorescence emission intensity of tyrosine residues, occurred at free calcium concentration ranging between 0.15 and 1.5 microM. Magnesium did not mimic the calcium effect. The results suggest that the 70-kDa protein possesses both high-affinity sites and selectivity for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

• Moncman CL, Peng I, Winkelmann DA
• Actin filament structure probed with monoclonal antibodies.
• Cell Motil Cytoskeleton. 1993; 25: 73-86
• Display abstract

• The interaction of two monoclonal antibodies (mAbs) with actin has been characterized to map the epitopes defined by these mAbs and to determine the accessibility of these sites in the actin filament (F-actin). Both mAbs react specifically with actin in radioimmunoassays and Western blot assays, and by immunoprecipitation. The location of the epitopes within the primary structure of actin has been determined using limited proteolysis of actin and Western blots, or using immunoprecipitation of truncated actin fragments synthesized in a cell free translation assay. Both mAbs bind to the C-terminal fragment of actin (residues 68-375) produced by chymotrypsin cleavage. One epitope is further localized to a 9.9 kD peptide corresponding to residues 5-93. Therefore, the epitope defined by this mAb (2G11.4) lies between residues Lys68 and Glu93 of actin. The location of the other epitope was determined by immunoprecipitation of actin fragments synthesized in vitro. Removal of residues 356-365 from the C-terminus of actin completely abolished the binding of mAb 4E3.adl. Therefore, this mAb defines an epitope that involves residues between Trp356 and Ala365. The accessibility of these epitopes in native F-actin was determined with solution binding assays and characterized by immunoelectron microscopy. Monoclonal antibody 4E3.adl binds strongly to filaments, resulting in bundling or decoration of F-actin depending on the valency of the mAb, and indicating that the epitope is readily accessible in F-actin. In contrast, mAb 2G11.4 disrupts F-actin structure, resulting in the formation of an amorphous immunoprecipitate. These results place constraints on models of actin filament structure.

• Hazes B et al.
• Crystal structure of deoxygenated Limulus polyphemus subunit II hemocyanin at 2.18 A resolution: clues for a mechanism for allosteric regulation.
• Protein Sci. 1993; 2: 597-619
• Display abstract

• The crystal structure of Limulus polyphemus subunit type II hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A. Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine N epsilon atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N delta atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5 degrees with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 degrees rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.

• McLaughlin PJ, Gooch JT, Mannherz HG, Weeds AG
• Structure of gelsolin segment 1-actin complex and the mechanism of filament severing.
• Nature. 1993; 364: 685-92
• Display abstract

• The structure of the segment 1 domain of gelsolin, a protein that fragments actin filaments in cells, is reported in complex with actin. Segment 1 binds monomer using an apolar patch rimmed by hydrogen bonds in a cleft between actin domains. On the actin filament model it binds tangentially, disrupting only those contacts between adjacent subunits in one helical strand. The segment 1 fold is general for all segments of the gelsolin family because the conserved residues form the core of the structure. It also provides a basis for understanding the origin of an amyloidosis caused by a gelsolin variant.

• Heintzelman MB, Frankel SA, Artavanis-Tsakonas S, Mooseker MS
• Cloning of a secretory gelsolin from Drosophila melanogaster.
• J Mol Biol. 1993; 230: 709-16
• Display abstract

• Gelsolin is an actin-binding protein with the abilities to sever and cap the barbed end of actin filaments and to promote the assembly of monomeric actin. It has been identified in vertebrates both as a cytoplasmic protein and as a protein secreted into the blood plasma. Here we report the nucleic acid sequence of the full-length complementary DNA for a secretory form of gelsolin from Drosophila. The deduced amino acid sequence of 790 residues (M(r) = 87,669) contains a predicted signal peptide of 20 amino acid residues. Comparison of the Drosophila gelsolin sequence with other members of the gelsolin family of actin-binding proteins reveals the characteristic segmental repeat structure found in this class of proteins. A 42% identity is observed between Drosophila secretory gelsolin and human plasma gelsolin when their primary structures are compared. Northern blots resolve a single 3000 base message in third instar Drosophila larvae, a message that appears to be encoded by a single gene located at 82A, B on the right arm of the third chromosome.

• Weigt C, Gaertner A, Wegner A, Korte H, Meyer HE
• Occurrence of an actin-inserting domain in tensin.
• J Mol Biol. 1992; 227: 593-5
• Display abstract

• We have previously described a protein called "insertin" that binds strongly to barbed ends of actin filaments and permits polymerization of actin filaments by insertion of actin monomers between the barbed ends and barbed end-bound insertin. We determined the amino acid sequence of insertin and found that the primary structure of insertin is almost identical to amino acid residues 862 to 1212 of the actin-binding protein tensin.

• Vandekerckhove J, Vancompernolle K
• Structural relationships of actin-binding proteins.
• Curr Opin Cell Biol. 1992; 4: 36-42
• Display abstract

• The sequences of a large number of actin-binding proteins have been compared. These findings, together with the results of protein-chemical analysis, peptide synthesis and site-directed and deletion mutagenesis, have led to the assignment of actin-binding sites. Within these segments, small actin-binding motifs have been delineated. Most actin-binding proteins interact with actin subdomain-1 but our analyses reveal neither primary nor secondary structure homology among these proteins, suggesting that actin binding does not follow simple structural principles.

• Namba Y, Ito M, Zu Y, Shigesada K, Maruyama K
• Human T cell L-plastin bundles actin filaments in a calcium-dependent manner.
• J Biochem (Tokyo). 1992; 112: 503-7
• Display abstract

• The amino acid sequences deduced from cDNA analyses revealed that human leucocyte L-plastin phosphorylated in response to interleukin 1, 2 closely resembles a chicken intestinal microvilli protein, fimbrin, that bundles actin filaments [de Arruda et al. (1990) J. Cell Biol. 111, 1069-1079]. In the present work, it was observed that unphosphorylated L-plastin isolated from human T cells bundled F-actin just as fimbrin does. L-Plastin acted on T cell beta-actin, but hardly acted on muscle alpha-actin or chicken gizzard gamma-actin, whereas fimbrin bundled muscle alpha-actin. Unlike fimbrin, L-plastin's actin-bundling action was strictly calcium-dependent: the bundles were formed at pCa 7, but not at pCa 6. Under suitable conditions, approximately one molecule of L-plastin bound to 8 molecules of actin monomer in the actin filament.

• Hori K, Morita F
• Actin-actin contact: inhibition of actin-polymerization by subdomain 4 peptide fragments.
• J Biochem (Tokyo). 1992; 112: 401-8
• Display abstract

• F-Actin was digested with alpha-chymotrypsin in 6 M urea, and two peptide fragments from subdomain 4 of actin molecule [Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes K.C. (1990) Nature 347, 37-44] were purified by reverse-phase HPLC and Sephadex G-50 gel filtration. The peptide fragments were identified as segments from Arg-177 to Tyr-198 (2.6-kDa peptide) and from Ser-199 to Tyr-279 (9.1-kDa peptide). Their effects on actin polymerization induced by 50 or 100 mM KCl were studied by measuring the increase in viscosity by the falling ball method. The 2.6-kDa peptide decreased the rate of actin polymerization and increased the critical concentration for the polymerization. Based on the atomic model of the actin filament [Holmes, K.C., Popp, D., Gebhard, W., & Kabsch, W. (1990) Nature 347, 44-49], the peptide is presumed to bind to the barbed end of the actin filament and inhibit the polymerization. By assuming that the peptide affected the rate of association of the actin monomer to the end of the actin filament, well-fitting curves for the polymerization kinetics were calculated. Computer-assisted results indicated that the dissociation constant of the 2.6-kDa peptide for F-actin is 200 to 260 microM. In contrast, the 9.1-kDa peptide only slightly inhibited actin polymerization. These results suggest that the actin-actin interface in the region between Arg-177 and Tyr-198 has a stronger interaction than those between Ser-199 and Tyr-279. The amino acid sequence L-T-D-Y-L present in the 2.6-kDa segment is homologous to a common sequence in the F-actin capping domain of various actin-binding proteins.

• Schneider R
• The human protooncogene ret: a communicative cadherin?
• Trends Biochem Sci. 1992; 17: 468-9

• Vancompernolle K, Goethals M, Huet C, Louvard D, Vandekerckhove J
• G- to F-actin modulation by a single amino acid substitution in the actin binding site of actobindin and thymosin beta 4.
• EMBO J. 1992; 11: 4739-46
• Display abstract

• The actin binding sites of actobindin and thymosin beta 4, two small polypeptides that inhibit actin polymerization by interacting with monomeric actin, have been localized using peptide mimetics. Both sites are functionally similar and extend over 20 residues and are located in the NH2-terminus of the polypeptides. They can be dissected into two functional entities: a conserved hexapeptide motif (LKHAET or LKKTET), which forms the major contact site through electrostatic interactions with actin, and a non-conserved NH2-terminal segment preceding the motif, which exerts the inhibitory activity on actin polymerization probably by steric hindrance. The introduction of a glutamic acid at the third position in the motif, creating LKEAET or LKETET sequences, which are similar to those found in some F-actin binding proteins, converts the peptide's inhibitory phenotype into an F-actin stimulatory property. These results allow the proposal of a simple model for G- to F-actin modulation.

• de Arruda MV, Bazari H, Wallek M, Matsudaira P
• An actin footprint on villin. Single site substitutions in a cluster of basic residues inhibit the actin severing but not capping activity of villin.
• J Biol Chem. 1992; 267: 13079-85
• Display abstract

• Villin is a multidomain protein that severs, caps, and bundles actin filaments. We employed a chemical modification/cleavage strategy to identify residues whose chemical reactivities are reduced when villin is complexed with actin. We found that actin protects 3 methionine residues, Met125, Met379, and Met711 from oxidation by N-chlorosuccinimide. Because Met125 lies within the actin-severing domain of villin (44T), we probed this region for actin binding sites using a series of overlapping peptides each with an additional cysteine residue at their C terminus. Each peptide, as a disulfide-bonded dimer, was examined for actin cross-linking activity by electron microscopy and light scattering. Our results with M3R suggest this region contains an F-actin binding site and are consistent with proteolysis and deletion mutagenesis studies of gelsolin. Single substitution of the basic residues modulated actin severing but not capping activity of 44T. Circular dichroism and protease digestions did not detect alterations in secondary structure or conformational changes in the mutants, although some are cleaved more rapidly, thereby suggesting a change in the packing of the domains. Our results highlight that basic residues comprise part of the F-actin binding site that is involved in the actin severing activity of villin.

• Friederich E et al.
• An actin-binding site containing a conserved motif of charged amino acid residues is essential for the morphogenic effect of villin.
• Cell. 1992; 70: 81-92
• Display abstract

• The actin-binding protein villin induces microvillus growth and reorganization of the cytoskeleton in cells that do not normally produce this protein. Transfection of mutagenized villin cDNAs into CV-1 cells was used to show that a conserved, COOH-terminally located cluster of charged amino acid residues (KKEK) is crucial for the morphogenic activity of villin in vivo. In vitro experiments with a 22 amino acid synthetic peptide corresponding to this region of villin provide evidence that this motif is part of an F-actin-binding site that induces G-actin to polymerize. Chemical cross-linking of actin to this peptide, the effects of amino acid substitutions in peptides, and the behavior of villin variants further corroborate the participation of the KKEK sequence in actin contacts.

• Jongstra-Bilen J, Janmey PA, Hartwig JH, Galea S, Jongstra J
• The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain.
• J Cell Biol. 1992; 118: 1443-53
• Display abstract

• The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.

• Hosoya H, Kobayashi R, Tsukita S, Matsumura F
• Ca(2+)-regulated actin and phospholipid binding protein (68 kD-protein) from bovine liver: identification as a homologue for annexin VI and intracellular localization.
• Cell Motil Cytoskeleton. 1992; 22: 200-10
• Display abstract

• An F-actin binding protein was purified from bovine liver by means of DNase I affinity, hydroxylapatite and DEAE-cellulose column chromatographies. It consisted of a single polypeptide chain having an apparent molecular weight of 68,000 with a Stokes radius of 35 A. Electron microscopy of rotary shadowed specimens showed that the 68 kD protein is a globular protein. This protein showed a higher affinity for F-actin in the presence of Ca2+ than in its absence, which is opposite to the actin-binding property shown by nonmuscle alpha-actinin or fimbrin. The 68 kD protein had no F-actin severing and capping activity. Interestingly, the 68 kD protein was found to aggregate liposomes at micromolar Ca2+ concentrations. Immunoblot analysis and partial protein sequence data identified the 68 kD protein as an annexin VI (p68) homologue. Immunocytochemical studies showed that the 68 kD protein was localized along stress fibers as well as membrane ruffles, microspikes and focal contacts, raising the possibility that annexin VI may contribute to control membrane-microfilament interaction in the cell.

• Way M, Pope B, Weeds AG
• Evidence for functional homology in the F-actin binding domains of gelsolin and alpha-actinin: implications for the requirements of severing and capping.
• J Cell Biol. 1992; 119: 835-42
• Display abstract

• The F-actin binding domains of gelsolin and alpha-actinin compete for the same site on actin filaments with similar binding affinities. Both contain tandem repeats of approximately 125 amino acids, the first of which is shown to contain the actin-binding site. We have replaced the F-actin binding domain in the NH2-terminal half of gelsolin by that of alpha-actinin. The hybrid severs filaments almost as efficiently as does gelsolin or its NH2-terminal half, but unlike the latter, requires calcium ions. The hybrid binds two actin monomers and caps the barbed ends of filaments in the presence or absence of calcium. The cap produced by the hybrid binds with lower affinity than that of gelsolin and is not stable: It dissociates from filament ends with a half life of approximately 15 min. Although there is no extended sequence homology between these two different F-actin binding domains, our experiments show that they are functionally equivalent and provide new insights into the mechanism of microfilament severing.

• Mannherz HG
• Crystallization of actin in complex with actin-binding proteins.
• J Biol Chem. 1992; 267: 11661-4

• Eichinger L, Schleicher M
• Characterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein.
• Biochemistry. 1992; 31: 4779-87
• Display abstract

• Severin is a Ca(2+)-activated actin-binding protein that nucleates actin assembly and severs and caps the fast growing ends of actin filaments. It consists of three highly conserved domains. To investigate the domain structure of severin, we constructed genetically the N-terminal domain 1, the middle domain 2, and the tandem domains 2 + 3. Their interaction with actin, Ca2+, and lipids was characterized. Domain 1 contains the F-actin capping and a Ca(2+)-binding site [Eichinger, L., Noegel, A. A., & Schleicher, M. (1991) J. Cell Biol. 112, 665-676]. Binding of domain 2 to actin filaments was Ca(2+)-dependent and saturated at a 1:1 molar ratio. In the presence of Ca2+, about 1.5 mol of domains 2 + 3 bound per mole of F-actin subunit. Scatchard analysis gave a Kd of 18 microM for the interaction of domain 2 with F-actin subunits and a Kd of 1.6 microM for domains 2 + 3. Low-shear viscometry, electron microscopy, and low-speed sedimentation assays showed that domains 2 + 3 induced bundling of actin filaments. The influence of PIP2 micelles on the different activities of severin was assayed using native severin and N- and C-terminally truncated fragments. Severin contains at least two PIP2-binding sites since the activities of the two nonoverlapping severin fragments domain 1 and domains 2 + 3 were inhibited by PIP2. The specificity of severin-phospholipid interaction was investigated by studying the regulation of native severin by PIP2 and other pure or mixed phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

• Chia CP, Hitt AL, Luna EJ
• Direct binding of F-actin to ponticulin, an integral plasma membrane glycoprotein.
• Cell Motil Cytoskeleton. 1991; 18: 164-79
• Display abstract

• We have developed an 125I-labeled F-actin blot overlay assay for the identification of F-actin-binding proteins after transfer to nitrocellulose from SDS-polyacrylamide gels. Two major F-actin-binding proteins from Dictyostelium discoideum, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F-actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin-binding glycoprotein from D. discoideum plasma membranes (Wuestehube, L.J., and Luna, E.J., [1987]: J. Cell Biol. 105:1741-1751). The binding of F-actin to ponticulin on blots is specific because unlabeled F-actin competes with 125I-labeled F-actin and because G-actin does not bind. Nitrocellulose-bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F-actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, 125-I-labeled F-actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2-D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F-actin in proportion to their abundance. Thus the actin-binding activies of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin-binding activity of nitrocellulose-bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The 125I-labeled F-actin blot overlay assay also may enable us to identify F-actin-binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.

• Way M, Pope B, Weeds A
• Molecular biology of actin binding proteins: evidence for a common structural domain in the F-actin binding sites of gelsolin and alpha-actinin.
• J Cell Sci Suppl. 1991; 14: 91-4
• Display abstract

• We review the impact of molecular biology on actin binding proteins, in particular on sequence relationships and expression of clones to dissect properties in vitro. Significant homologies exist between proteins in each class, but we propose, in addition, that common structural features exist between the F-actin binding sites of severing and cross-linking proteins.

• Cook WJ, Ealick SE, Babu YS, Cox JA, Vijay-Kumar S
• Three-dimensional structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor.
• J Biol Chem. 1991; 266: 652-6
• Display abstract

• The three-dimensional structure of a sarcoplasmic Ca2(+)-binding protein from the sandworm Nereis diversicolor has been determined at 3.0 A resolution using multiple isomorphous replacement techniques. The NH2-terminal half of the molecule contains one variant Ca2(+)-binding domain with a novel helix-loop-helix conformation and one Ca2(+)-binding domain that is no longer functional because of amino acid changes. The overall conformation of this pair of domains is different from any previously described Ca2(+)-binding protein. The COOH-terminal half of the protein contains two Ca2(+)-binding domains with the usual helix-loop-helix configuration and is similar to calmodulin and troponin C. Unlike calmodulin or troponin C, there is no exposed alpha-helix connecting the two halves of the molecule, so the overall structure is much more compact.

• Fechheimer M, Murdock D, Carney M, Glover CV
• Isolation and sequencing of cDNA clones encoding the Dictyostelium discoideum 30,000-dalton actin-bundling protein.
• J Biol Chem. 1991; 266: 2883-9
• Display abstract

• The Dictyostelium 30,000-dalton protein is a calcium-regulated actin filament-bundling protein which has been suggested to contribute to the structure and reorganization of filopodia and pseudopodia accompanying cell movements. cDNAs encoding this protein were isolated using antibody and oligonucleotide probes to screen cDNA libraries in phage lambda. The sequence of the cDNA predicts a protein of 295 amino acids with a molecular weight of 33,355. The sequence reveals two EF-hand calcium-binding regions that provide a structural explanation for calcium regulation of the activity of this protein. The putative calcium-binding region of the 30,000-dalton protein has similarity to sequences of other calcium-regulated actin-binding proteins such as alpha-actin and fimbrin. One region of the sequence with similarity to both Dictyostelium gelation factor (ABP 120) and fructose bisphosphate aldolase is a potential actin-binding sequence. A highly charged region of the protein is similar to a sequence in human cytovillin that is repeated eight times in chicken gizzard caldesmon. No strong homology to previously identified actin-binding sequences of other actin-binding proteins is apparent. Results from Southern blot experiments indicate that the 30,000-dalton protein is encoded by a single gene in the Dictyostelium genome.

• Adams AE, Botstein D, Drubin DG
• Requirement of yeast fimbrin for actin organization and morphogenesis in vivo.
• Nature. 1991; 354: 404-8
• Display abstract

• The SAC6 gene was found by suppression of a yeast actin mutation. Its protein product, Sac6p (previously referred to as ABP67), was independently isolated by actin-filament affinity chromatography and colocalizes with actin in vivo. Thus Sac6p binds to actin in vitro, and functionally associates with actin structures involved in the development and maintenance of cell polarity in vivo. We report here that Sac6p is an actin-filament bundling protein 43% identical in amino-acid sequence to the vertebrate bundling protein fimbrin. This yeast fimbrin homologue contains two putative actin-binding regions homologous to domains of dystrophin, beta-spectrin, filamin, actin-gelation protein and alpha-actinin. Mutants lacking Sac6p do not form normal actin structures and are defective in morphogenesis. These findings demonstrate an in vivo role for the well-documented biochemical interaction between fimbrin and actin.

• Dubreuil RR
• Structure and evolution of the actin crosslinking proteins.
• Bioessays. 1991; 13: 219-26
• Display abstract

• The actin crosslinking proteins exhibit marked diversity in size and shape and crosslink actin filaments in different ways. Amino acid sequence analysis of many of these proteins has provided clues to the origin of their diversity. Spectrin, alpha-actinin, ABP-120, ABP-280, fimbrin, and dystrophin share a homologous sequence segment that is implicated as the common actin binding domain. The remainder of each protein consists of repetitive and non-repetitive sequence segments that have been shuffled and multiplied in evolution to produce a variety of proteins that are related in function and in composition, but that differ significantly in structure.

• Weis WI, Kahn R, Fourme R, Drickamer K, Hendrickson WA
• Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing.
• Science. 1991; 254: 1608-15
• Display abstract

• Calcium-dependent (C-type) animal lectins participate in many cell surface recognition events mediated by protein-carbohydrate interactions. The C-type lectin family includes cell adhesion molecules, endocytic receptors, and extracellular matrix proteins. Mammalian mannose-binding proteins are C-type lectins that function in antibody-independent host defense against pathogens. The crystal structure of the carbohydrate-recognition domain of a rat mannose-binding protein, determined as the holmium-substituted complex by multiwavelength anomalous dispersion (MAD) phasing, reveals an unusual fold consisting of two distinct regions, one of which contains extensive nonregular secondary structure stabilized by two holmium ions. The structure explains the conservation of 32 residues in all C-type carbohydrate-recognition domains, suggesting that the fold seen here is common to these domains. The strong anomalous scattering observed at the Ho LIII edge demonstrates that traditional heavy atom complexes will be generally amenable to the MAD phasing method.

• Bresnick AR, Janmey PA, Condeelis J
• Evidence that a 27-residue sequence is the actin-binding site of ABP-120.
• J Biol Chem. 1991; 266: 12989-93
• Display abstract

• Proteolysis experiments of ABP-120 from Dictyostelium discoideum have previously demonstrated that removal of residues 89-115 from a tryptic peptide which retains actin binding activity, abolishes actin binding (Bresnick, A. R., Warren, V., and Condeelis, J. (1990) J. Biol. Chem. 265, 9236-9240). Antibodies made against a synthetic peptide of this 27-amino acid sequence (27-mer) specifically immunoprecipitate native ABP-120 from Dictyostelium high speed supernatants, demonstrating that the 27-mer sequence is on the surface of the molecule as expected for an active site. ABP-120 is inhibited in its binding to F-actin by Fab' fragments of the anti-27-mer IgG. Half-maximal inhibition occurs at an approximate molar ratio of 7 Fab' fragments/ABP-120 monomer. Viscoelastic measurements indicate that ABP-120 forms fewer cross-links with F-actin in the presence of the 27-mer synthetic peptide than in its absence. In F-actin cosedimentation assays, the binding of ABP-120 to actin is inhibited by the 27-mer synthetic peptide. Furthermore, the 27-mer synthetic peptide cosediments with F-actin, whereas a control hydrophobic peptide and a synthetic peptide of residues 69-88 of ABP-120 do not cosediment with F-actin. These observations suggest a direct involvement of the 27-mer sequence in the actin binding activity of ABP-120.

• Matsudaira P
• Modular organization of actin crosslinking proteins.
• Trends Biochem Sci. 1991; 16: 87-92
• Display abstract

• A family of actin-crosslinking proteins share a conserved 125 residue sequence that lies within a 250 residue actin-binding domain. This domain is combined with spacer segments consisting of a variable number of repeated alpha-helical or beta-sheet motifs and other functional domains, which generate proteins that differ in their ability to form actin bundles or networks and to associate with the plasma membrane. These functional domains are not in other actin-crosslinking proteins, one of which is elongation factor 1a (EF-1a) suggesting there are several pathways for the evolution of actin-crosslinking function.

• Zu YL et al.
• 65-kilodalton protein phosphorylated by interleukin 2 stimulation bears two putative actin-binding sites and two calcium-binding sites.
• Biochemistry. 1990; 29: 8319-24
• Display abstract

• We have previously characterized a 65-kilodalton protein (p65) as an interleukin 2 stimulated phosphoprotein in human T cells and showed that three endopeptide sequences of p65 are present in the sequence of l-plastin [Zu et al. (1990) Biochemistry 29, 1055-1062]. In this paper, we present the complete primary structure of p65 based on the cDNA isolated from a human T lymphocyte (KUT-2) cDNA library. Analysis of p65 sequences and the amino acid composition of cleaved p65 N-terminal peptide indicated that the deduced p65 amino acid sequence exactly coincides with that of l-plastin over the C-terminal 580 residues [Lin et al. (1988) Mol. Cell. Biol. 8, 4659-4668] and has a 57-residue extension at the N-terminus to l-plastin. Computer-assisted structural analysis revealed that p65 is a multidomain molecule involving at least three intriguing functional domains: two putative calcium-binding sites along the N-terminal 80 amino acid residues; a putative calmodulin-binding site following the calcium-binding region; and two tandem repeats of putative actin-binding domains in its middle and C-terminal parts, each containing approximately 240 amino acid residues. These results suggest that p65 belongs to actin-binding proteins.

• Sri Widada J et al.
• Identification of the calmodulin binding domain of alpha-fodrin and implications for folding.
• Biochimie. 1990; 72: 19-24
• Display abstract

• A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.

• Way M, Pope B, Gooch J, Hawkins M, Weeds AG
• Identification of a region in segment 1 of gelsolin critical for actin binding.
• EMBO J. 1990; 9: 4103-9
• Display abstract

• The actin severing and capping protein gelsolin contains three distinct actin binding sites. The smallest actin binding domain of approximately 15,000 Mr was originally obtained by limited proteolysis and it corresponds to the first of six repeating segments contained in the gelsolin sequence. We have expressed this domain (here termed segment 1 or N150 to define its amino acid length) in Escherichia coli, together with a series of smaller mutants truncated at either N- or C-terminal ends, in an attempt to localize residues critical of actin binding. Limited truncation of segment 1 by 11 residues at its N-terminal end has no observable effect on actin binding, but on removal of a further eight residues, actin binding is totally eliminated. Although this loss of actin binding may reflect ablation of critical residues, we cannot rule out the possibility that removal of these residues adversely affects the folding of the polypeptide chain during renaturation. Truncation at the C-terminus of segment 1 has a progressive effect on actin binding. Unlike intact segment 1, which shows no calcium sensitivity of actin binding within the resolution of our assays, a mutant with 19 residues deleted from its C-terminus shows unchanged affinity for actin in the presence of calcium, but approximately 100-fold weaker binding in its absence. Removal of an additional five residues from the C-terminus produces a mutant that binds actin only in calcium. Further limited truncation results in progressively weaker calcium dependent binding and all binding is eliminated when a total of 29 residues has been removed. Although none of the expressed proteins on their own binds calcium, 45Ca is trapped in the complexes, including the complex between actin and segment 1 itself. These results highlight a region close to the C-terminus of segment 1 that is essential for actin binding and demonstrate that calcium plays an important role in the high affinity actin binding by this domain of gelsolin.

• Ahmed FR, Przybylska M, Rose DR, Birnbaum GI, Pippy ME, MacManus JP
• Structure of oncomodulin refined at 1.85 A resolution. An example of extensive molecular aggregation via Ca2+.
• J Mol Biol. 1990; 216: 127-40
• Display abstract

• The crystal structure of oncomodulin, a 12,000 Mr protein isolated from rat tumours, has been determined by molecular replacement using the carp parvalbumin structure as a starting model. Refinement was performed by cycles of molecular fitting and restrained least-squares, using area-detector intensity data to 1.85 A resolution. For the 5770 reflections in the range 6.0 to 1.85 A, which were used in the refinement, the crystallographic R-factor is 0.166. The refined model includes residues 2 to 108, three Ca2+ and 87 water molecules per oncomodulin molecule. The oncomodulin backbone is closely related to that of parvalbumin; however, some differences are found after a least-squares fit of the two backbones, with root-mean-square (r.m.s.) deviations of 1 to 2 A in residues 2 to 6, 59 to 61 of the CD loop, 87, 90 and 108. The overall r.m.s. deviation of the backbone residues 5 to 108 is 0.62 A. Each of the two Ca2+ atoms that are bound to the CD and EF loops is co-ordinated to seven oxygen atoms, including one water molecule. The third Ca2+ is also seven-co-ordinated, to five oxygen atoms belonging to three different oncomodulin molecules and to two water molecules which form hydrogen bonds to a fourth oncomodulin; thus, this intermolecular Ca2+ and its equivalents interlink the molecules into zigzag layers normal to the b axis with a spacing of b/2 or 32.14 A. No such extensive molecular aggregation has been reported for any of the related Ca-binding regulatory proteins of the troponin-C family studied thus far. The Ca-O distances in all three polyhedra are in the range 2.07 A to 2.64 A, indicating tightly bound Ca polyhedra.

• DasGupta G, White J, Phillips M, Bulinski JC, Reisler E
• Immunochemical probing of the N-terminal segment on actin: the polymerization reaction.
• Biochemistry. 1990; 29: 3319-24
• Display abstract

• The N-terminal segment of actin contains a cluster of acidic residues which are implicated in macromolecular interactions of this protein. In this work, the interrelationship between the N-terminal segment and the polymerization of actin was studied by using affinity-purified antibodies directed against the first seven N-terminal residues on alpha-skeletal actin (S alpha N). The Fab fragments of these antibodies showed equal affinities for G- and F-actin while the bivalent IgG bound preferentially to the polymerized actin. As monitored by pyrene fluorescence measurements, the binding of Fab to G-actin did not alter the kinetics of the MgCl2-induced polymerization; IgG accelerated this reaction considerably. Consistent with these observations, the binding of Fab to F-actin did not change its morphological appearance in electron micrographs and had no effect on the stability and the rate of dissociation of actin filaments. These results are discussed in terms of their implications to the spatial relationship between the N-terminal segment and the rest of the molecule and the context of the polymerization reaction of actin in vitro and in vivo.

• Bresnick AR, Warren V, Condeelis J
• Identification of a short sequence essential for actin binding by Dictyostelium ABP-120.
• J Biol Chem. 1990; 265: 9236-40
• Display abstract

• Tryptic digestion of ABP-120, an actin cross-linking protein from Dictyostelium discoideum, generates a ladder of peptides differing in molecular mass by 13,000 daltons, indicating a structural repeat within the molecule. A number of peptides bind actin with the smallest having a molecular mass of 17,000 daltons (T17). Our sedimentation assays also show that a peptide of 14,000 daltons does not bind actin. Using the full-length cDNA sequence (Noegel, A., Rapp, S., Lottspeich, F., Schleicher, M., and Stewart, M. (1989) J. Cell Biol. 109, 607-618) and protein sequencing techniques, we have determined that T17 begins at residue 89 while T14 begins at residue 116. Therefore we have localized 27 amino acids which are essential for actin binding activity. This region is at the end of the molecule, distal from the repetitive beta-sheet region predicted from the cDNA sequence, and displays high sequence identity with regions in the N termini of ABP/filamin, dystrophin, beta-spectrin, and alpha-actinin.

• Haltia M et al.
• Amyloid protein in familial amyloidosis (Finnish type) is homologous to gelsolin, an actin-binding protein.
• Biochem Biophys Res Commun. 1990; 167: 927-32
• Display abstract

• Familial amyloidosis, Finnish type, is clinically characterized by cranial neuropathy and lattice corneal dystrophy. It is an autosomal dominant form of systemic amyloidosis with small deposits of congophilic material occurring in most tissues, particularly in association with blood vessel walls and basement membranes. Amyloid fibrils were extracted from the kidney of patient VUO, and rabbit antiserum raised against the 12 kDa purified amyloid subunit displayed strong immunohistochemical reactivity with the amyloid deposits. The amino terminal sequence of this 12 kDa amyloid protein (ATEVPVSWESFNNGD) showed homology with gelsolin (or actin depolymerizing factor), a 93 kDa plasma protein. The amyloid peptide is a degradation product, starting at position 173, of the gelsolin molecule.

• Vancompernolle K, Gimona M, Herzog M, Van Damme J, Vandekerckhove J, Small V
• Isolation and sequence of a tropomyosin-binding fragment of turkey gizzard calponin.
• FEBS Lett. 1990; 274: 146-50
• Display abstract

• Limited chymotryptic cleavage of turkey gizzard calponin yields a 13 kDa fragment which could be purified by its ability to bind to Sepharose-immobilized tropomyosin. This 13 kD polypeptide is shown to be derived from a 22 kDa fragment. Complete amino acid sequence analysis of the 13 kD and 22 kD fragments reveals high homology with the formerly characterized smooth muscle-specific protein SM22 alpha (Pearlstone, J.R., Weber, M., Lees-Miller, J.P., Carpenter, M.R. and Smillie L.B., 1987, J. Biol. Chem. 262, 5985-5991) and the product of gene mp20 of Drosophila (Ayme-Southqate, A., Lasko, P., French, C, and Pardue, M.L. [(1989) J. Cell Biol. 108, 521-531]. Futhermore we recognize sequence elements of a putative actin-binding domain of alpha-actinin, the calpactin I or p 36 sequence, and a consensus motif present in the repeats of the gene product of the candidate unc-87 gene of C. elegans (S.D. Goetinck and R.H. Waterston, personal communication).

• Huber R, Schneider M, Mayr I, Romisch J, Paques EP
• The calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution. Implications for membrane binding and calcium channel activity.
• FEBS Lett. 1990; 275: 15-21
• Display abstract

• Crystal structure analysis and refinement at 2.0 A resolution of a rhombohedral crystal form of human annexin V at high calcium concentration revealed a domain motion compared to the previously analysed hexagonal crystal form. Five calcium ions were located on the convex face of the molecule. Three strongly bound calciums are liganded at protruding interhelical loops and Asp or Glu residues in homologous positions in repeats I, II and IV. Five proteinaceous oxygens and one solvent molecule form the coordination polyhedron in each case. The unoccupied seventh site is suggested as the phospholipid headgroup binding site. Two more weakly bound sites were identified by lanthanum labelling. The structural features suggest that annexin V attaches with its convex face to membranes by specific calcium mediated interactions with at least three phospholipids. The adjacent membrane bilayer may thus become locally disordered and permeable to allow calcium inflow through the central polar channel of the molecule.

• Berchtold MW
• Structure and expression of genes encoding the three-domain Ca2+-binding proteins parvalbumin and oncomodulin.
• Biochim Biophys Acta. 1989; 1009: 201-15

• Tellam RL, Morton DJ, Clarke FM
• A common theme in the amino acid sequences of actin and many actin-binding proteins?
• Trends Biochem Sci. 1989; 14: 130-3
• Display abstract

• The amino acid sequences of several actin regulatory proteins have recently been determined. Do these proteins function by mimicking actin-actin interaction sites?

• Vandekerckhove J
• Structural principles of actin-binding proteins.
• Curr Opin Cell Biol. 1989; 1: 15-22

• Strynadka NC, James MN
• Crystal structures of the helix-loop-helix calcium-binding proteins.
• Annu Rev Biochem. 1989; 58: 951-98

• Booth KS, Kimura S, Lee HC, Ikeda-Saito M, Caughey WS
• Bovine myeloperoxidase and lactoperoxidase each contain a high affinity site for calcium.
• Biochem Biophys Res Commun. 1989; 160: 897-902
• Display abstract

• Both bovine myeloperoxidase and lactoperoxidase contain one calcium per iron with no other metal present in significant amount. Calcium is bound with high affinity and is removed upon exposure to 6 M guanidine hydrochloride/EGTA which results in precipitation of protein. Computer amino acid sequence analyses of human myeloperoxidase reveal two plausible calcium binding sites. This is the first evidence for the presence of calcium in these peroxidases.

• Sri Widada J et al.
• Cloning and deletion mutagenesis using direct protein-protein interaction on an expression vector. Identification of the calmodulin binding domain of alpha-fodrin.
• J Mol Biol. 1989; 205: 455-8
• Display abstract

• We have screened a lambda gt11 library, constructed with mouse macrophage cDNA, in order to isolate clones that code for calmodulin binding proteins. We have developed a new approach for this purpose using radioactive calmodulin (produced by genetic engineering) to detect fusion proteins that interact with this protein with high affinity. A cDNA clone that codes for mouse macrophage fodrin was isolated, sequenced and identified. By deleting part of the sequence the calmodulin binding domain was located on the fodrin sequence. The site is situated on repeat 11 of fodrin and probably on the extra arm of this repeat. The method we developed is widely applicable to site-directed mutagenesis of interacting proteins.

• Sekharudu YC, Sundaralingam M
• A structure-function relationship for the calcium affinities of regulatory proteins containing 'EF-hand' pairs.
• Protein Eng. 1988; 2: 139-46
• Display abstract

• Using a series of homologous calcium-binding proteins, a quantitative structure-activity relationship (QSAR), log(1/Kd) = -18.986 - 1.6278(X1) + 0.7981(X2) + 0.2312(X3), has been established, which relates the calcium-binding affinities (1/Kd) of the regulatory proteins with (i) the net ligand charge (X1) of the two calcium binding loops, (ii) the hydrophobicity (X2) of the beta-sheet segment of the loops and (iii) the hydrophobicity (X3) of the four 'EF-hand' helices. It is found that the binding affinities are influenced by the 'EF-hand' pair rather than the individual 'EF-hands'. The QSAR, in addition to explaining satisfactorily the large variation in the observed calcium affinities, can predict the affinities of the 'EF-hand' pairs in other proteins from the amino acid sequence and can also account for the changes in the affinities caused by substitution in the hydrophobic and/or metal-coordinating residues. Thus, this relationship can be employed in protein design and engineering. The method is potentially useful in the development of similar relationships for the binding of other proteins to substrates, inhibitors, drugs and co-factors.

• Gribskov M, Homyak M, Edenfield J, Eisenberg D
• Profile scanning for three-dimensional structural patterns in protein sequences.
• Comput Appl Biosci. 1988; 4: 61-6
• Display abstract

• Profile analysis measures the similarity between a target sequence and a group of aligned sequences (the probe). The probe sequences are used to produce a position-specific scoring table (the profile) that can be aligned with any sequence (the target) using standard dynamic programming methods. We are developing a library of profiles, each describing a different structural motif. This allows any target sequence to be rapidly scanned for the presence of structural motifs. Levels of significance for the comparison of target sequences with the profile are determined in advance, permitting an objective decision to be made as to whether a protein is likely to possess a structural motif.

• Petersen TE
• The amino-terminal domain of thrombomodulin and pancreatic stone protein are homologous with lectins.
• FEBS Lett. 1988; 231: 51-3
• Display abstract

• Amino acid sequence alignment of the amino-terminal of thrombomodulin and pancreatic stone protein with the hepatic asialoglycoprotein receptor shows that these proteins are homologous. From the known disulfide bridge pattern of other proteins belonging to the same family two disulfide bonds can be predicted. The homology raises the question whether the amino-terminal part of thrombomodulin and the pancreatic protein binds carbohydrate or perhaps like tetranectin have a specific affinity for other proteins.

• Andre E, Lottspeich F, Schleicher M, Noegel A
• Severin, gelsolin, and villin share a homologous sequence in regions presumed to contain F-actin severing domains.
• J Biol Chem. 1988; 263: 722-7
• Display abstract

• cDNA clones encoding the actin filament severing protein severin from Dictyostelium discoideum were isolated from a cDNA library in lambda gt 11 using monoclonal antibodies. Comparison of the deduced amino acid sequence with the sequence of a severin peptide indicated that the complete coding region of severin is contained in the isolated clones. Severin, a 39.9-kDa protein, is encoded by one gene in D. discoideum. An mRNA of approximately 1.4 kilobases is present throughout the developmental cycle of D. discoideum. The amino acid sequence of severin contains a region highly homologous to a conserved sequence in villin and gelsolin, two proteins of similar function isolated from vertebrates. This homologous region is believed to participate in the actin filament severing activity of these proteins. Comparison of the severin sequence to the entire gelsolin sequence shows remarkable homologies pointing to a common origin from an ancestral gene from which gelsolin has been derived by a duplication.

• Schleicher M, Andre E, Hartmann H, Noegel AA
• Actin-binding proteins are conserved from slime molds to man.
• Dev Genet. 1988; 9: 521-30
• Display abstract

• DNA clones encoding the actin-binding proteins alpha-actinin and severin from Dictyostelium discoideum were isolated and sequenced. Comparisons of the deduced amino acid sequences with proteins from other species showed striking similarities at distinct regions. The F-actin cross-linking molecule alpha-actinin carries two characteristic EF-hand structures highly homologous to the Ca2+-binding loops of proteins from the calmodulin superfamily. An N-terminal region that is conserved in alpha-actinin from D. discoideum and vertebrates is also related to parts of the dystrophin sequence and might represent the F-actin binding site. Severin, gelsolin, villin, and fragmin share homologous sequences that are believed to participate in the severing activity of these proteins.

• Patthy L
• Homology of human pancreatic stone protein with animal lectins.
• Biochem J. 1988; 253: 309-11

• Bazari WL, Matsudaira P, Wallek M, Smeal T, Jakes R, Ahmed Y
• Villin sequence and peptide map identify six homologous domains.
• Proc Natl Acad Sci U S A. 1988; 85: 4986-90
• Display abstract

• Site-specific proteases and antisera to the amino terminus of villin have been used to show that villin is organized into seven protease-resistant domains. Six are contained in the amino-terminal Mr 87,000 villin core, a Ca2+-regulated actin-severing fragment, whereas the carboxyl-terminal domain includes the villin "headpiece," a fragment involved in bundling of actin filaments. Ca2+ inhibits proteolytic cleavage between domains in the amino-terminal half of villin. The protein sequence of villin deduced from a single cDNA clone contains a conserved sequence that is repeated six times and is found in each domain of the villin core. The conserved repeats are found in other actin-severing proteins but not in the villin headpiece. Our results suggest that actin-severing proteins are organized around a common Mr 14,000-17,000 domain.

• Arpin M et al.
• Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity.
• J Cell Biol. 1988; 107: 1759-66
• Display abstract

• Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation. The amino acid sequence deduced from the cDNA sequence revealed that human villin is composed of three domains. The first two domains appear as the result of a duplication: their structural organization is similar. We can then define a basic unit in which a slightly hydrophilic motif is followed by three hydrophobic motifs, similar between themselves and regularly spaced. The duplicated domain is highly homologous to three other actin-severing proteins and this basic structure represents the whole molecule in severin and fragmin, while two basic units compose gelsolin. The third domain which is carboxy terminal is villin specific: it is unique among actin modulating proteins so far known. It could account for its actin-binding properties (dual regulation by calcium of severing and bundling activities). We propose that it may also be related to the subcellular localization of villin in different epithelial cell types.

• Franke WW
• Homology of a conserved sequence in the tail domain of intermediate filament proteins with the loop region of calcium binding proteins.
• Cell Biol Int Rep. 1987; 11: 831-831

• Ampe C, Vandekerckhove J
• The F-actin capping proteins of Physarum polycephalum: cap42(a) is very similar, if not identical, to fragmin and is structurally and functionally very homologous to gelsolin; cap42(b) is Physarum actin.
• EMBO J. 1987; 6: 4149-57
• Display abstract

• We have carried out a primary structure analysis of the F-actin capping proteins of Physarum polycephalum. Cap42(b) was completely sequenced and was found to be identical with Physarum actin. Approximately 88% of the sequence of cap42(a) was determined. Cap42(a) and fragmin were found to be identical by amino acid composition, isoelectric point, mol. wt, elution time on reversed-phase chromatography and amino acid sequence of their tryptic peptides. The available sequence of cap42(a) is greater than 36% homologous with the NH2-terminal 42-kd domain of human gelsolin. A highly homologous region of 16 amino acids is also shared between cap42(a), gelsolin and the Acanthamoeba profilins. Cap42(a) binds two actin molecules in a similar way to gelsolin suggesting a mechanism of F-actin modulation that has been conserved during evolution.

• Taylor WR, Geisow MJ
• Predicted structure for the calcium-dependent membrane-binding proteins p35, p36, and p32.
• Protein Eng. 1987; 1: 183-7
• Display abstract

• A new family of proteins (annexins) that bind to membranes at micromolar free Ca2+ has been recognized. Its members include an EGF-receptor kinase substrate (p35), a retroviral tyrosine kinase substrate (p36), the liver protein endonexin (p32) and an electric ray protein, calelectrin. Each protein contains four sequence repeats with a further 2-fold internal homology. Using the predicted secondary structure and pattern of conserved hydrophobic residues in each repeat, we have built a three-dimensional model that is largely isostructural with the known molecular conformation of bovine intestinal calcium-binding protein. The final (energy-refined) model had a core formed from the conserved hydrophobic residues. It differed from ICaBP principally in the length of the two Ca2+-binding loops with only one loop being able to bind. The model suggests a mechanism for interaction of these new Ca2+-binding proteins with phospholipid bilayers.

• Wuestehube LJ, Luna EJ
• F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes.
• J Cell Biol. 1987; 105: 1741-51
• Display abstract

• F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge.

• Kretsinger RH
• Calcium coordination and the calmodulin fold: divergent versus convergent evolution.
• Cold Spring Harb Symp Quant Biol. 1987; 52: 499-510

• Kwiatkowski DJ, Stossel TP, Orkin SH, Mole JE, Colten HR, Yin HL
• Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain.
• Nature. 1986; 323: 455-8
• Display abstract

• Gelsolin is representative of a class of actin-modulating proteins found in lower eukaryotes to mammals, which sever actin filaments. Gelsolin found in the cytoplasm of cells is functionally similar to a mammalian plasma protein of similar size, originally called ADF or brevin. Human plasma and rabbit macrophage gelsolins differ by the presence of a 25-amino-acid residue extension on plasma gelsolin which appears to account for the difference in relative molecular mass (Mr) between the proteins as assessed by SDS-polyacrylamide gel electrophoresis (PAGE), 93,000 (93K) and 90K, respectively. Here we report the isolation of full-length human plasma gelsolin complementary DNA clones from a HepG2 library. The inferred amino-acid sequence reveals the presence of a signal peptide, a long tandem repeat that matches the actin-binding domains of gelsolin, a tetrapeptide present in actin and extended regions of identical sequence with rabbit macrophage gelsolin. Southern blot analysis indicates that a single gene in the haploid genome encodes both protein forms.

• Tamkun JW et al.
• Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin.
• Cell. 1986; 46: 271-82
• Display abstract

• We describe the isolation, characterization, and sequence of cDNA clones encoding one subunit of the complex of membrane glycoproteins that forms part of the transmembrane connection between the extracellular matrix and the cytoskeleton. The cDNA sequence encodes a polypeptide of 89 kd that has features strongly suggesting the presence of a large N-terminal extracellular domain, a single transmembrane segment, and a small C-terminal cytoplasmic domain. The extracellular domain contains a threefold repeat of a novel 40 residue cysteine-rich segment, and the cytoplasmic domain contains a tyrosine residue that is a potential site for phosphorylation by tyrosine kinases. We propose the name integrin for this protein complex to denote its role as an integral membrane complex involved in the transmembrane association between the extracellular matrix and the cytoskeleton.

• Geisow MJ
• Common domain structure of Ca2+ and lipid-binding proteins.
• FEBS Lett. 1986; 203: 99-103
• Display abstract

• The phospholipase A2 inhibitor, lipocortin, shares common sequences with three abundant Ca2+-regulated membrane binding proteins of unknown function which are present in many cell and tissue types. A two-domain model for the structure of lipocortin is described and it is suggested that the new Ca2+-regulated proteins each contain at least one lipocortin domain. The structural and biochemical properties of each protein indicate that they all directly interact with phospholipids. Potential sites of interaction with the lipocortin domain are identified on the basis of homology with phospholipid transfer proteins and phospholipase A2.

• Bader MF, Trifaro JM, Langley OK, Thierse D, Aunis D
• Secretory cell actin-binding proteins: identification of a gelsolin-like protein in chromaffin cells.
• J Cell Biol. 1986; 102: 636-46
• Display abstract

• Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.

• Kwiatkowski DJ, Janmey PA, Mole JE, Yin HL
• Isolation and properties of two actin-binding domains in gelsolin.
• J Biol Chem. 1985; 260: 15232-8
• Display abstract

• Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.

• Matsudaira P, Jakes R, Walker JE
• A gelsolin-like Ca2+-dependent actin-binding domain in villin.
• Nature. 1985; 315: 248-50
• Display abstract

• Villin is an actin-binding protein of relative molecular mass (Mr) 95,000 found in the core bundle of microfilaments in brush border microvilli from intestine. In physiological calcium concentrations (less than 1 microM), villin crosslinks actin filaments into bundles. However, in free calcium concentrations (greater than 1 microM), villin severs actin filaments into short pieces. To understand how villin can sever and bundle actin filaments, we are studying the molecular basis of villin-actin binding interactions by identifying important actin-binding domains in villin. Here, we report the purification and preliminary characterization of a 44,000-Mr fragment of villin which contains a calcium-dependent actin-severing activity. In addition, the partial amino-acid sequence from the amino terminus of this fragment reveals homology with a 16-residue region near the amino terminus of gelsolin, an actin-severing protein found in many cells and sera. The sequence homology suggests a common structural basis for the calcium-regulated actin-severing properties shared by villin and gelsolin.

• Stossel TP
• Contribution of actin to the structure of the cytoplasmic matrix.
• J Cell Biol. 1984; 99: 1521-1521

• Sutoh K, Iwane M, Matsuzaki F, Kikuchi M, Ikai A
• Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia.
• J Cell Biol. 1984; 98: 1611-8
• Display abstract

• A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.

• Hauschka PV, Carr SA, Biemann K
• Primary structure of monkey osteocalcin.
• Biochemistry. 1982; 21: 638-42
• Display abstract

• The complete 49-residue amino acid sequence of osteocalcin from the old world monkey Macaca fascicularis has been determined by efficient combination of gas chromatography-mass spectrometry and Edman techniques. This vitamin K dependent protein of bone matrix contains three gamma-carboxyglutamic acid residues at positions 17, 21, and 24, as well as a disulfide-bonded loop (23--29). Features of the sequence which apparently are required for the binding of Ca2+ have been strongly conserved throughout evolution.

• Weeds A
• Actin-binding proteins--regulators of cell architecture and motility.
• Nature. 1982; 296: 811-6
• Display abstract

• Numerous actin-binding proteins from a variety of cell types have been described. Here I attempt to correlate the properties and functions of some of these. Three major classes have been identified: (1) cross-linking proteins which form filament bundles or isotropic gels; (2) proteins which cap filament ends and nucleate the polymerization of G-actin (many of these also sever actin filaments); (3) proteins which bind to G-actin and stabilize the monomer pool. Some of the proteins described here combine the properties of more than one class and the activities of many of them are regulated by changes in Ca2+ ion concentration.

• Glenney JR Jr, Geisler N, Kaulfus P, Weber K
• Demonstration of at least two different actin-binding sites in villin, a calcium-regulated modulator of F-actin organization.
• J Biol Chem. 1981; 256: 8156-61
• Display abstract

• Villin, one of the calcium regulated modulator proteins of F-actin organization, restricts F-actin to short filaments in the presence of calcium and bundles F-actin in the absence of calcium. Limited in vitro proteolysis of villin generates, in addition to a large core fragment (apparent Mr = 90,000) previously described, a small headpiece (Mr = 8,500). The finding that the F-actin nucleation and severing activity of villin, but not its bundling activity, is retained by the core suggested that the headpiece may be directly involved in bundling. Headpiece has now been purified and characterized. It shows strong F-actin binding both in the presence and absence of calcium, leading to a final stoichiometry of 1 headpiece to 1 F-actin monomer. Headpiece also inhibits villin-induced F-actin bundling. Thus villin expresses at least two distinct actin-binding sites localized on separate functional domains. Protein sequence analysis documents that the core comprises the NH2-terminal portion of intact villin, whereas the headpiece covers the COOH-terminal 76 amino acids. We provide the amino acid sequence of the headpiece, which is currently the smallest F-actin binding peptide.

• Kretsinger RH
• Crystallographic studies of calmodulin and homologs.
• Ann N Y Acad Sci. 1980; 356: 14-9

© 2021 EMBL | Privacy Policy