NORMAL GENOMIC

• Frank AT et al.
• Structure and function of glycosylated tandem repeats from Candidaalbicans Als adhesins.
• Eukaryot Cell. 2010; 9: 405-14
• Display abstract

• Tandem repeat (TR) regions are common in yeast adhesins, but theirstructures are unknown, and their activities are poorly understood. TRregions in Candida albicans Als proteins are conserved glycosylated36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R.De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke,Eukaryot. Cell 5:1664-1673, 2006). Ab initio modeling with either Rosettaor LINUS generated consistent structures of three-stranded antiparallelbeta-sheet domains, whereas randomly shuffled sequences with the samecomposition generated various structures with consistently higherenergies. O- and N-glycosylation patterns showed that each TR domain hadexposed hydrophobic surfaces surrounded by glycosylation sites. Thesestructures are consistent with domain dimensions and stabilitymeasurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A.Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677-1682,2009) and with circular dichroism determination of secondary structure andthermal stability. Functional assays showed that the hydrophobic surfacesof TR domains supported binding to polystyrene surfaces and other TRdomains, leading to nonsaturable homophilic binding. The domain structuresare like "classic" subunit interaction surfaces and can explain previouslyobserved patterns of promiscuous interactions between TR domains in anyAls proteins or between TR domains and surfaces of other proteins.Together, the modeling techniques and the supporting data lead to anapproach that relates structure and function in many kinds of repeatdomains in fungal adhesins.

• Gilbert HE, Eaton JT, Hannan JP, Holers VM, Perkins SJ
• Solution structure of the complex between CR2 SCR 1-2 and C3d of humancomplement: an X-ray scattering and sedimentation modelling study.
• J Mol Biol. 2005; 346: 859-73
• Display abstract

• Complement receptor type 2 (CR2, CD21) forms a tight complex with C3d, afragment of C3, the major complement component. Previous crystalstructures of the C3d-CR2 SCR 1-2 complex and free CR2 SCR 1-2 showed thatthe two SCR domains of CR2 form contact with each other in a closedV-shaped structure. SCR 1 and SCR 2 are connected by an unusually longeight-residue linker peptide. Medium-resolution solution structures forCR2 SCR 1-2, C3d, and their complex were determined by X-ray scatteringand analytical ultracentrifugation. CR2 SCR 1-2 is monomeric. For CR2 SCR1-2, its radius of gyration R(G) of 2.12(+/-0.05) nm, its maximum lengthof 10nm and its sedimentation coefficient s20,w(o) of 1.40(+/-0.03) S donot agree with those calculated from the crystal structures, and insteadsuggest an open structure. Computer modelling of the CR2 SCR1-2 solutionstructure was based on the structural randomisation of the eight-residuelinker peptide joining SCR 1 and SCR 2 to give 9950 trial models.Comparisons with the X-ray scattering curve indicated that the mostfavoured arrangements for the two SCR domains corresponded to an openV-shaped structure with no contacts between the SCR domains. For C3d,X-ray scattering and sedimentation velocity experiments showed that itexists as a monomer-dimer equilibrium with a dissociation constant of 40microM. The X-ray scattering curve for monomeric C3d gave an R(G) value of1.95 nm, and this together with its s20,w(o) value of 3.17 S gave goodagreement with the monomeric C3d crystal structure. Modelling of the C3ddimer gave good agreements with its scattering and ultracentrifugationparameters. For the complex, scattering and ultracentrifugationexperiments showed that there was no dimerisation, indicating that the C3ddimerisation site was located close to the CR2 SCR 1-2 binding site. TheR(G) value of 2.44(+/-0.1) nm, its length of 9 nm and its s20,w(o) valueof 3.45(+/-0.01) S showed that its structure was not much more elongatedthan that of C3d. Calculations with 9950 models of CR2 SCR 1-2 bound toC3d through SCR 2 showed that SCR 1 formed an open V-shaped structure withSCR 2 and was capable of interacting with the surface of C3d. We concludethat the open V-shaped structures formed by CR2 SCR 1-2, both when freeand when bound to C3d, are optimal for the formation of a tight two-domaininteraction with its ligand C3d.

• Piao S et al.
• Crystal structure of a clip-domain serine protease and functional roles ofthe clip domains.
• EMBO J. 2005; 24: 4404-14
• Display abstract

• Clip-domain serine proteases (SPs) are the essential components ofextracellular signaling cascades in various biological processes,especially in embryonic development and the innate immune responses ofinvertebrates. They consist of a chymotrypsin-like SP domain and one ortwo clip domains at the N-terminus. Prophenoloxidase-activating factor(PPAF)-II, which belongs to the noncatalytic clip-domain SP family, isindispensable for the generation of the active phenoloxidase leading tomelanization, a major defense mechanism of insects. Here, the crystalstructure of PPAF-II reveals that the clip domain adopts a novel foldcontaining a central cleft, which is distinct from the structures ofdefensins with a similar arrangement of cysteine residues. Ensuing studiesdemonstrated that PPAF-II forms a homo-oligomer upon cleavage by theupstream protease and that the clip domain of PPAF-II functions as amodule for binding phenoloxidase through the central cleft, while the clipdomain of a catalytically active easter-type SP plays an essential role inthe rapid activation of its protease domain.

• Ohki I et al.
• Crystal structure of human lectin-like, oxidized low-density lipoproteinreceptor 1 ligand binding domain and its ligand recognition mode to OxLDL.
• Structure. 2005; 13: 905-17
• Display abstract

• Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, isthe major receptor for oxidized LDL (OxLDL) in endothelial cells. We havedetermined the crystal structure of the ligand binding domain of LOX-1,with a short stalk region connecting the domain to the membrane-spanningregion, as a homodimer linked by an interchain disulfide bond. In vivoassays with LOX-1 mutants revealed that the "basic spine," consisting oflinearly aligned arginine residues spanning over the dimer surface, isresponsible for ligand binding. Single amino acid substitution in thedimer interface caused a severe reduction in LOX-1 binding activity,suggesting that the correct dimer arrangement is crucial for binding toOxLDL. Based on the LDL model structure, possible binding modes of LOX-1to OxLDL are proposed.

• Wang G, Wylie GP, Twigg PD, Caspar DL, Murphy JR, Logan TM
• Solution structure and peptide binding studies of the C-terminal srchomology 3-like domain of the diphtheria toxin repressor protein.
• Proc Natl Acad Sci U S A. 1999; 96: 6119-24
• Display abstract

• The diphtheria toxin repressor (DtxR) is the best-characterized member ofa family of homologous proteins that regulate iron uptake and virulencegene expression in the Gram-positive bacteria. DtxR contains two domainsthat are separated by a short, unstructured linker. The N-terminal domainis structurally well-defined and is responsible for Fe2+ binding,dimerization, and DNA binding. The C-terminal domain adopts a fold similarto eukaryotic Src homology 3 domains, but the functional role of theC-terminal domain in repressor activity is unknown. The solution structureof the C-terminal domain, consisting of residues N130-L226 plus a13-residue N-terminal extension, has been determined by using NMRspectroscopy. Residues before A147 are highly mobile and adopt a randomcoil conformation, but residues A147-L226 form a single structured domainconsisting of five beta-strands and three helices arranged into apartially orthogonal, two-sheet beta-barrel, similar to the structureobserved in the crystalline Co2+ complex of full-length DtxR. Chemicalshift perturbation studies demonstrate that a proline-rich peptidecorresponding to residues R125-G139 of intact DtxR binds to the C-terminaldomain in a pocket formed by residues in beta-strands 2, 3, and 5, andhelix 3. Binding of the proline-rich peptide by the C-terminal domain ofDtxR presents an example of peptide binding by a prokaryotic Src homology3-like protein. The results of this study, combined with previous x-raystudies of intact DtxR, provide insights into a possible biologicalfunction of the C-terminal domain in regulating repressor activity.

• Paoloni-Giacobino A, Chen H, Peitsch MC, Rossier C, Antonarakis SE
• Cloning of the TMPRSS2 gene, which encodes a novel serine protease withtransmembrane, LDLRA, and SRCR domains and maps to 21q22.3.
• Genomics. 1997; 44: 309-20
• Display abstract

• To contribute to the development of the transcription map of humanchromosome 21 (HC21), we have used exon trapping from pools ofHC21-specific cosmids. Using selected trapped exons, we have identified anovel gene (named TMPRSS2) that encodes a multimeric protein with a serineprotease domain. The TMPRSS2 3.8-kb mRNA is expressed strongly in smallintestine and weakly in several other tissues. The full-length cDNAencodes a predicted protein of 492 amino acids that contains the followingdomains: (i) A serine protease domain (aa 255-492) of the S1 family thatprobably cleaves at Arg or Lys residues. (ii) An SRCR (scavenger receptorcysteine-rich) domain (aa 149-242) of group A (6 conserved Cys). This typeof domain is involved in the binding to other cell surface orextracellular molecules. (iii) An LDLRA (LDL receptor class A) domain (aa113-148). This type of domain forms a binding site for calcium. (iv) Apredicted transmembrane domain (aa 84-106). No typical signal peptide wasrecognized. The gene was mapped to 21q22.3 between markers ERG and D21S56in the same P1 as MX1. The physiological role of TMPRSS2 and itsinvolvement in trisomy 21 phenotypes or monogenic disorders that map toHC21 are unknown.

• Sunnerhagen M, Olah GA, Stenflo J, Forsen S, Drakenberg T, Trewhella J
• The relative orientation of Gla and EGF domains in coagulation factor X isaltered by Ca2+ binding to the first EGF domain. A combined NMR-smallangle X-ray scattering study.
• Biochemistry. 1996; 35: 11547-59
• Display abstract

• Coagulation factor X is a serine protease containing three noncatalyticdomains: an N-terminal gamma-carboxyglutamic acid (Gla)1 domain followedby two epidermal growth factor (EGF)-like domains. The isolated N-terminalEGF domain binds Ca2+ with a Kd of 10(-3) M. When linked to the Gladomain, however, its Ca2+ affinity is increased 10-fold. In this paper, wepresent the NMR solution structure of the factor X Gla-EGF domain pairwith Ca2+ bound to the EGF domain, as well as small angle X-ray scattering(SAXS) data on the Gla-EGF domain pair with and without Ca2+. Our resultsshow that Ca2+ binding to the EGF domain makes the Gla and EGF domainsfold toward each other using the Ca2+ site as a hinge. Presumably, asimilar mechanism may be responsible for alterations in the relativeorientation of protein domains in many other extracellular proteinscontaining EGF domains with the consensus for Ca2+ binding. The results ofthe NMR and SAXS measurements reported in this paper confirm our previousresult that the Gla domain is folded also in its apo state when linked tothe EGF domain [Sunnerhagen, M., et al. (1995) Nat. Struct. Biol. 2,504-509]. Finally, our study clearly demonstrates the powerful combinationof NMR and SAXS in the study of modular proteins, since this enablesreliable evaluation of both short-range (NMR) and long-range interactions(SAXS).

• Smith KF, Harrison RA, Perkins SJ
• Molecular modeling of the domain structure of C9 of human complement byneutron and X-ray solution scattering.
• Biochemistry. 1992; 31: 754-64
• Display abstract

• C9 is the most abundant component of the membrane attack complex of thecomplement system of immune defense. This is a typical mosaic protein withthrombospondin (TSR) and low density lipoprotein receptor (LDLr) domainsat its N-terminus and an epidermal growth factor-like (EGF) domain at itsC-terminus. Between these lies a perforin-like sequence. In order todefine the arrangement in solution of these four moieties in C9, high-fluxneutron and synchrotron X-ray solution scattering studies were carriedout. The neutron radius of gyration RG at infinite contrast is 3.33 nm,and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtainedby synchrotron X-ray scattering after allowance for radiation effects.Stuhrmann analyses showed that the neutron radial inhomogeneity ofscattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5)from the RXS data. These values are typical for soluble glycoproteins andshow no evidence for the existence of any large hydrophobic surfacepatches on free C9 that might form contacts with lipids. Indirecttransformation of the neutron and X-ray scattering curves into real spaceshowed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and thissuggests that the lengths of 7-8 nm deduced from previous electronmicroscopy studies in vacuo are underestimated. Molecular modeling of theC9 scattering curves utilized small spheres in the Debye equation, inwhich the analyses were constrained by the known volumes of the fourmoieties of C9 and the known sizes of the TSR and EGF-like domains. Themost likely models for C9 suggest that these four regions of C9 arearranged in a V-shaped structure, with an angle of 10 degrees between thetwo arms, each of length 11.1 nm. This structure has a more hydrophobiccharacter between the two arms. The scattering model is fully consistentwith hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamicmodels could be developed for C6, C7, C8, and C9 of complement. Such acompact structure is atypical of other multidomain complement proteins sofar studied by solution scattering and is fully compatible with mechanismsin which C9 is postulated, on activation, to undergo a drastic unfoldingof its domain structure and to expose a more hydrophobic surface which canbe embedded into lipid bilayers.