NORMAL GENOMIC

• Han B, Yang H, Wang QL, Chen W, Qian CW, Xiong S
• [Purification of Cyanovirin-N with antiviral activity and preparation as well as modification of its polyclonal antibody].
• Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2011; 27: 531-4
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• AIM: To purify the recombinant Cyanovirin-N (CVN) and determine its anti-influenza virus A (H1N1) activity, and to prepare the polyclonal antibody of CVN, purify it and label it with an enzyme for future application. METHODS: The recombinant CVN were rapidly purified by two rounds of Ni-NTA chelating chromatography intervened with a SUMO protease cleavage step. Anti-H1N1 activity of CVN was determined using cytopathogenic effect assay (CPE). The rabbits were immunized with the purified CVN and the antibody was identified by ELISA and Western blot. IgG was purified by ammonium sulfate precipitation followed by DEAE chromatography and the purified IgG was labeled by HRP. RESULTS: The purity of the obtained CVN protein which showed obvious Anti-H1N1 activity in vitro was higher than 95%.The polyclonal antibody of CVN was successfully produced in the rabbits and the results of ELISA and Western blot showed that the antiserum had high titer and high specificity. The purified antibody with a titer up to 1:6 400 was successfully obtained and the anti-CVN antibody-HRP conjugate was achieved after labeling the purified antibody with HRP. CONCLUSION: The purified antibody against CVN has been purified and further coniugated with HRP, with can be used for future research.

• Kittur FS, Yu HY, Bevan DR, Esen A
• Homolog of the maize beta-glucosidase aggregating factor from sorghum is a jacalin-related GalNAc-specific lectin but lacks protein aggregating activity.
• Glycobiology. 2009; 19: 277-87
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• Recently, we identified the maize beta-glucosidase aggregating factor (BGAF) as a jacalin-related lectin (JRL) and showed that its lectin domain is responsible for beta-glucosidase aggregation. By searching for BGAF homologs in sorghum, we identified and obtained an EST clone and determined its complete sequence. The predicted protein had the same modular structure as maize BGAF, shared 67% sequence identity with it, and revealed the presence of two potential carbohydrate-binding sites (GG...ATYLQ, site I and GG...GVVLD, site II). Maize BGAF1 is the only lectin from a class of modular JRLs containing an N-terminal dirigent and a C-terminal JRL domain, whose sugar specificity and beta-glucosidase aggregating activity have been studied in detail. We purified to homogeneity a BGAF homolog designated as SL (Sorghum lectin) from sorghum and expressed its recombinant version in Escherichia coli. The native protein had a molecular mass of 32 kD and was monomeric. Both native and recombinant SL-agglutinated rabbit erythrocytes, and inhibition assays indicated that SL is a GalNAc-specific lectin. Exchanging the GG...GVVLD motif in SL with that of maize BGAF1 (GG...GIAVT) had no effect on GalNAc-binding, whereas binding to Man was abolished. Substitution of Thr(293) and Gln(296) in site I to corresponding residues (Val(294) and Asp(297)) of maize BGAF1 resulted in the loss of GalNAc-binding, indicating that site I is responsible for generating GalNAc specificity in SL. Gel-shift and pull-down assays after incubating SL with maize and sorghum beta-glucosidases showed no evidence of interaction nor were any SL-protein complexes detected in sorghum tissue extracts, suggesting that the sorghum homolog does not participate in protein-protein interactions.

• Wittenbrink M, Spreu J, Steinle A
• Differential NKG2D binding to highly related human NKG2D ligands ULBP2 and RAET1G is determined by a single amino acid in the alpha2 domain.
• Eur J Immunol. 2009; 39: 1642-51
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• The activating NK cell receptor NKG2D binds to numerous stress-induced cell surface glycoproteins with MHC class I-related ectodomains. In humans, NKG2D ligands (NKG2DL) are members of the MHC-encoded MIC and non-MHC-encoded ULBP families of proteins. The redundancy of NKG2DL raises questions about unique features associated with individual NKG2DL. The ULBP family member RAET1G contains an ectodomain highly related to ULBP2, but is unique by virtue of an extended cytoplasmic domain. Since RAET1G is poorly characterized, we studied expression and functional interactions of RAET1G in comparison to ULBP2. RAET1G transcripts were detected in most human tissues with an overall expression pattern similar to ULBP2. However, although ULBP2 strongly binds both NKG2D and the immunoevasive human cytomegalovirus glycoprotein UL16, RAET1G only weakly interacts with NKG2D and does not bind UL16. Differential binding capacities of the two highly related ectodomains are mainly due to a substitution of a conserved amino acid in the alpha2 domain of RAET1G. In functional terms, the reduced apparent avidity of RAET1G for NKG2D results in a less-efficient NKG2D down-regulation and NK degranulation. Altogether, RAET1G, like ULBP2, appears broadly expressed, but exhibits a lower apparent avidity for NKG2D due to a mutation in the center of the MHC-like fold.

• Fromme R, Katiliene Z, Fromme P, Ghirlanda G
• Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 A resolution.
• Protein Sci. 2008; 17: 939-44
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• Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 A resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 A resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.

• Chen DE, Podell S, Sauer JD, Swanson MS, Saier MH Jr
• The phagosomal nutrient transporter (Pht) family.
• Microbiology. 2008; 154: 42-53
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• Phagosomal transporters (Phts), required for intracellular growth of Legionella pneumophila, comprise a novel family of multispanning alpha-helical proteins within the major facilitator superfamily (MFS). The members of this family derive exclusively from bacteria. Multiple paralogues are present in a restricted group of Alpha- and Gammaproteobacteria, but single members were also found in Chlamydia and Cyanobacteria. Their protein sequences were aligned, yielding a phylogenetic tree showing the relations of the proteins to each other. Topological analyses revealed a probable 12 alpha-helical transmembrane segment (TMS) topology. Motif identification and statistical analyses provided convincing evidence that these proteins arose from a six TMS precursor by intragenic duplication. The phylogenetic tree revealed some potential orthologous relationships, suggestive of common function. However, several probable examples of lateral transfer of the encoding genetic material between bacteria were identified and analysed. The Pht family most closely resembles a smaller MFS family (the UMF9 family) with no functionally characterized members. However, the UMF9 family occurs in a broader range of prokaryotic organism types, including Archaea. These two families differ in that organisms bearing members of the Pht family often have numerous paralogues, whereas organisms bearing members of the UMF9 family never have more than two. This work serves to characterize two novel families within the MFS and provides compelling evidence for horizontal transfer of some of the family members.

• Bersch B et al.
• Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge.
• J Mol Biol. 2008; 380: 386-403
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• The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10(-5) M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-beta fold formed of two beta-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal beta-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal beta-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal beta-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-beta fold and the methionine-rich Cu(I)-binding site.

• Chitayat S et al.
• Three-dimensional structure of a putative non-cellulosomal cohesin module from a Clostridium perfringens family 84 glycoside hydrolase.
• J Mol Biol. 2008; 375: 20-8
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• The genomes of myonecrotic strains of Clostridium perfringens encode a large number of secreted glycoside hydrolases. The activities of these enzymes are consistent with degradation of the mucosal layer of the human gastrointestinal tract, glycosaminoglycans and other cellular glycans found throughout the body. In many cases this is thought to aid in the propagation of the major toxins produced by C. perfringens. One such example is the family 84 glycoside hydrolases, which contains five C. perfringens members (CpGH84A-E), each displaying a unique modular architecture. The smallest and most extensively studied member, CpGH84C, comprises an N-terminal catalytic domain with beta-N-acetylglucosaminidase activity, a family 32 carbohydrate-binding module, a family 82 X-module (X82) of unknown function, and a fibronectin type-III-like module. Here we present the structure of the X82 module from CpGH84C, determined by both NMR spectroscopy and X-ray crystallography. CpGH84C X82 adopts a jell-roll fold comprising two beta-sheets formed by nine beta-strands. CpGH84C X82 displays distant amino acid sequence identity yet close structural similarity to the cohesin modules of cellulolytic anaerobic bacteria. Cohesin modules are responsible for the assembly of numerous hydrolytic enzymes in a cellulose-degrading multi-enzyme complex, termed the cellulosome, through a high-affinity interaction with the calcium-binding dockerin module. A planar surface is located on the face of the CpGH84 X82 structure that corresponds to the dockerin-binding region of cellulolytic cohesin modules and has the approximate dimensions to accommodate a dockerin module. The presence of cohesin-like X82 modules in glycoside hydrolases of C. perfringens is an indication that the formation of novel X82-dockerin mediated multi-enzyme complexes, with potential roles in pathogenesis, is possible.

• Mishra AK, Gangwani L, Davis RJ, Lambright DG
• Structural insights into the interaction of the evolutionarily conserved ZPR1 domain tandem with eukaryotic EF1A, receptors, and SMN complexes.
• Proc Natl Acad Sci U S A. 2007; 104: 13930-5
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• Eukaryotic genomes encode a zinc finger protein (ZPR1) with tandem ZPR1 domains. In response to growth stimuli, ZPR1 assembles into complexes with eukaryotic translation elongation factor 1A (eEF1A) and the survival motor neurons protein. To gain insight into the structural mechanisms underlying the essential function of ZPR1 in diverse organisms, we determined the crystal structure of a ZPR1 domain tandem and characterized the interaction with eEF1A. The ZPR1 domain consists of an elongation initiation factor 2-like zinc finger and a double-stranded beta helix with a helical hairpin insertion. ZPR1 binds preferentially to GDP-bound eEF1A but does not directly influence the kinetics of nucleotide exchange or GTP hydrolysis. However, ZPR1 efficiently displaces the exchange factor eEF1Balpha from preformed nucleotide-free complexes, suggesting that it may function as a negative regulator of eEF1A activation. Structure-based mutational and complementation analyses reveal a conserved binding epitope for eEF1A that is required for normal cell growth, proliferation, and cell cycle progression. Structural differences between the ZPR1 domains contribute to the observed functional divergence and provide evidence for distinct modalities of interaction with eEF1A and survival motor neuron complexes.

• Fromme R et al.
• A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity.
• Biochemistry. 2007; 46: 9199-207
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• Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.

• Milla MA, Townsend J, Chang IF, Cushman JC
• The Arabidopsis AtDi19 gene family encodes a novel type of Cys2/His2 zinc-finger protein implicated in ABA-independent dehydration, high-salinity stress and light signaling pathways.
• Plant Mol Biol. 2006; 61: 13-30
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• The AtDi19 (drought-induced) gene family encodes seven hydrophilic proteins that contain two atypical Cys2/His2 (C2H2) zinc finger-like domains that are evolutionarily well-conserved within angiosperms suggesting a conserved and important function. Five of the seven Arabidopsis AtDi19-related:DsRed2 fusion proteins exhibited speckled patterns of localization within the nucleus as shown by transient expression analysis in Arabidopsis protoplasts. In contrast, AtDi19-2:DsRed2 was present in the nucleus and cytoplasm, whereas AtDi19-4:DsRed2 was localized to the nuclear periphery. mRNA expression studies showed that AtDi19 genes are ubiquitously expressed in Arabidopsis tissues, although some differences were observed. In seedlings, RT-PCR analyses showed that AtDi19-1 and AtDi19-3 steady-state transcript amounts were rapidly induced by dehydration, whereas transcript amounts for AtDi19-2 and AtDi19-4 increased in response to high-salt stress. In addition, the mRNA abundance of all the AtDi19-related gene family members was not regulated by ABA. These data, taken together, suggest that several AtDi19-related gene family members may function in ABA-independent, dehydration and salinity stress signaling pathways. However, they may also be regulated by other abiotic stimuli. AtDi19-7, for example, has been implicated in regulating light signaling and responses. Finally, we show that most AtDi19-related proteins are phosphorylated in vitro by calcium-dependent protein kinases suggesting that this post-translational modification may be important for regulating the function of this novel protein family.

• Kinch LN, Grishin NV
• Longin-like folds identified in CHiPS and DUF254 proteins: vesicle trafficking complexes conserved in eukaryotic evolution.
• Protein Sci. 2006; 15: 2669-74
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• Eukaryotic protein trafficking pathways require specific transfer of cargo vesicles to different target organelles. A number of vesicle trafficking and membrane fusion components participate in this process, including various tethering factor complexes that interact with small GTPases prior to SNARE-mediated vesicle fusion. In Saccharomyces cerevisiae a protein complex of Mon1 and Ccz1 functions with the small GTPase Ypt7 to mediate vesicle trafficking to the vacuole. Mon1 belongs to DUF254 found in a diverse range of eukaryotic genomes, while Ccz1 includes a CHiPS domain that is also present in a known human protein trafficking disorder gene (HPS-4). The present work identifies the CHiPS domain and a sequence region from another trafficking disorder gene (HPS-1) as homologs of an N-terminal domain from DUF254. This link establishes the evolutionary conservation of a protein complex (HPS-1/HPS-4) that functions similarly to Mon1/Ccz1 in vesicle trafficking to lysosome-related organelles of diverse eukaryotic species. Furthermore, the newly identified DUF254 domain is a distant homolog of the mu-adaptin longin domain found in clathrin adapter protein (AP) complexes of known structure that function to localize cargo protein to specific organelles. In support of this fold assignment, known longin domains such as the AP complex sigma-adaptin, the synaptobrevin N-terminal domains sec22 and Ykt6, and the srx domain of the signal recognition particle receptor also regulate vesicle trafficking pathways by mediating SNARE fusion, recognizing specialized compartments, and interacting with small GTPases that resemble Ypt7.

• Barrientos LG, Matei E, Lasala F, Delgado R, Gronenborn AM
• Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition.
• Protein Eng Des Sel. 2006; 19: 525-35
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• The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the trimannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.

• Xie B et al.
• Identification of the fibroblast growth factor (FGF)-interacting domain in a secreted FGF-binding protein by phage display.
• J Biol Chem. 2006; 281: 1137-44
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• Fibroblast growth factor-binding proteins (FGF-BP) are secreted carrier proteins that release fibroblast growth factors (FGFs) from the extracellular matrix storage and thus enhance FGF activity. Here we have mapped the interaction domain between human FGF-BP1 and FGF-2. For this, we generated T7 phage display libraries of N-terminally and C-terminally truncated FGF-BP1 fragments that were then panned against immobilized FGF-2. From this panning, a C-terminal fragment of FGF-BP1 (amino acids 193-234) was identified as the minimum binding domain for FGF. As a recombinant protein, this C-terminal fragment binds to FGF-2 and enhances FGF-2-induced signaling in NIH-3T3 fibroblasts and GM7373 endothelial cells, as well as mitogenesis and chemotaxis of NIH-3T3 cells. The FGF interaction domain in FGF-BP1 is distinct from the heparin-binding domain (amino acids 110-143), and homology modeling supports the notion of a distinct domain in the C terminus that is conserved across different species. This domain also contains conserved positioning of cysteine residues with the Cys-214/Cys-222 positions in the human protein predicted to participate in disulfide bridge formation. Phage display of a C214A mutation of FGF-BP1 reduced binding to FGF-2, indicating the functional significance of this disulfide bond. We concluded that the FGF interaction domain is contained in the C terminus of FGF-BP1.

• Takahashi A, Inokoshi J, Chiba H, Omura S, Tanaka H
• Essential regions for antiviral activities of actinohivin, a sugar-binding anti-human immunodeficiency virus protein from an actinomycete.
• Arch Biochem Biophys. 2005; 437: 233-40
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• Actinohivin (AH) is a potent anti-human immunodeficiency virus (HIV) protein that consists of highly conserved three-tandem repeats (segments 1, 2, and 3). The molecular target of AH in its anti-HIV activity is high-mannose-type saccharide chains of HIV gp120. This article deals with sequence requirements for the anti-HIV activity of AH. The deleted or substituted DNAs encoding AH or His-AH were prepared using mutagenic oligonucleotide primers in PCR. The mutant constructs were expressed in Escherichia coli, and the activities of the recombinant protein products were examined by a syncytium-formation assay system that mimics anti-HIV activity. The single segment mutant His-AHs showed no anti-syncytium-formation activity, but the mutant His-AHs, which consists of 2 or 3 segments, retained reduced activities. His-AH(6-114) dramatically reduced the anti-syncytium-formation activity to that of His-AH(36-114) or His-AH(I5A). Furthermore, His-AH(Q33A), His-AH(Q71A), and His-AH(Q109A) in which glutamine residues were substituted into alanine showed reduced activities of 1/20, 1/10, and 1/30, respectively, in anti-syncytium formation compared with His-AH. These results indicate that three segments of AH are necessary for potent anti-syncytium-formation activity-that is, for potent anti-HIV activity and the cooperated involvement of each segment of AH increased the AH-gp120 interaction.

• Behr M, Hoch M
• Identification of the novel evolutionary conserved obstructor multigene family in invertebrates.
• FEBS Lett. 2005; 579: 6827-33
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• Insects have evolved chitin-containing structures such as the cuticle or peritrophic membranes that serve to protect their bodies against the hostile environment. The specific mechanisms by which these structures are produced, are mostly unknown. We have identified a novel multigene family, the obstructor family, which encodes ten putatively secreted chitin-binding proteins that are characterized by a stereotype arrangement of a N-terminal signaling peptide and 3 chitin-binding-domains. Gene expression studies in Drosophila melanogaster embryos demonstrate that obstructor family members are expressed in cuticle forming tissues. Using computational and phylogenetic analysis, we show that obstructor genes represent an evolutionary conserved multigene family in invertebrates.

• Margulis CJ
• Computational study of the dynamics of mannose disaccharides free in solution and bound to the potent anti-HIV virucidal protein cyanovirin.
• J Phys Chem B. 2005; 109: 3639-47
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• In this paper, we present a computational study of the dynamics of the potent anti-HIV virucidal protein cyanovirin in complex with mannose disaccharides. Recently, it has been experimentally demonstrated that cyanovirin binds mannose oligomers on the surface of glycoprotein gp120. gp120, a protein on the surface of the HIV virus, is key in the process of viral docking and transfer of genetic material into human cells. Cyanovirin prevents the transfer of viral RNA into human cells. In this study, we found that, among all residues that show nuclear Overhauser effects in the solution NMR experiments, residues Glu41 and Arg76 appear to interact with the sugar at the high-affinity binding site through stronger Coulombic interactions. In particular, Arg76 participates in a dynamical mechanism that caps and locks the sugar once it is bound to the protein. We also studied the distribution of glycosidic torsional angles of mannose disaccharides in solution and compared it with those when bound at the high- and low-affinity sites of the protein. Throughout our 20 ns simulations, we find that the sugar bound to the high-affinity site preserves the most favorable conformation in solution while the sugar bound at the low-affinity site does not. The sugar at the low-affinity site can adopt both conformations, but we find it most predominantly on the one that is least probable for the free sugar in solution. We also carried out a detailed study of the interactions between the disaccharides and different amino acids as well as between the disaccharide and the solvent at both binding locations.

• Sandstrom C, Berteau O, Gemma E, Oscarson S, Kenne L, Gronenborn AM
• Atomic mapping of the interactions between the antiviral agent cyanovirin-N and oligomannosides by saturation-transfer difference NMR.
• Biochemistry. 2004; 43: 13926-31
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• The minimum oligosaccharide structure required for binding to the potent HIV-inactivating protein cyanovirin-N (CV-N) was determined by saturation-transfer difference (STD) NMR spectroscopy. Despite the low molecular mass of the protein (11 kDa), STD-NMR spectroscopy allowed the precise atomic mapping of the interactions between CV-N and various di- and trimannosides, substructures of Man-9, the predominant oligosaccharide on the HIV viral surface glycoprotein gp120. Contacts with mannosides containing the terminal Manalpha(1-->2)Manalpha unit of Man-9 were observed, while (1-->3)- and (1-6)-linked di- and trimannosides showed no interactions, demonstrating that the terminal Manalpha(1-->2)Manalpha structure plays a key role in the interaction. Precise epitope mapping revealed that, for Manalpha(1-->2)ManalphaOMe, Manalpha(1-->2)Manalpha(1-->3)ManalphaOMe, and Manalpha(1-->2)Manalpha(1-->6)ManalphaOMe, the protein is in close contact with H2, H3, and H4 of the nonreducing terminal mannose unit. In contrast, the STD-NMR spectrum of the CV-N/trisaccharide Manalpha(1-->2)Manalpha(1-->2)ManalphaOMe complex was markedly different, with resonances on all sugar units displaying equal enhancements, suggesting that CV-N is able to discriminate between the three structurally related trisaccharides.

• Botos I, Wlodawer A
• Cyanovirin-N: a sugar-binding antiviral protein with a new twist.
• Cell Mol Life Sci. 2003; 60: 277-87
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• Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium Nostoc ellipsosporum, is a highly potent virucidal agent that has generated interest as a lead natural product for the prevention and chemotherapy of human immunodeficiency virus infection. The antiviral activity of CV-N is mediated through specific, high-affinity interactions with the viral surface envelope glycoproteins. A number of structures of wild-type, mutant and sequence-shuffled CV-N have been solved by nuclear magnetic resonance and crystallography, showing that the protein exists as either a quasi-symmetric two-domain monomer or a domain-swapped dimer. Structures of several complexes of CV-N with oligosaccharides help in explaining the unique mode of high-affinity binding of these molecules to both forms of CV-N.

• Mori T, Barrientos LG, Han Z, Gronenborn AM, Turpin JA, Boyd MR
• Functional homologs of cyanovirin-N amenable to mass production in prokaryotic and eukaryotic hosts.
• Protein Expr Purif. 2002; 26: 42-9
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• Cyanovirin-N (CV-N) is under development as a topical (vaginal or rectal) microbicide to prevent sexual transmission of human immunodeficiency virus (HIV), and an economically feasible means for very large-scale production of the protein is an urgent priority. We observed that N-glycosylation of CV-N in yeast eliminated the anti-HIV activity, and that dimeric forms and aggregates of CV-N occurred under certain conditions, potentially complicating the efficient, large-scale manufacture of pure monomeric CV-N. We therefore expressed and tested CV-N homologs in which the glycosylation-susceptible Asn residue at position 30 was replaced with Ala, Gln, or Val, and/or the Pro at position 51 was replaced by Gly to eliminate potential conformational heterogeneity. All homologs exhibited anti-HIV activity comparable to wild-type CV-N, and the Pro51Gly homologs were significantly more stable proteins. These glycosylation-resistant, functional cyanovirins should be amenable to large-scale production either in bacteria or in eukaryotic hosts.

• Bewley CA
• Rapid validation of the overall structure of an internal domain-swapped mutant of the anti-HIV protein cyanovirin-N using residual dipolar couplings.
• J Am Chem Soc. 2001; 123: 1014-5