Solution and crystal structures of a sugar binding site mutant of cyanovirin-N: no evidence of domain swapping.

The cyanobacterial lectin Cyanovirin-N (CV-N) exhibits antiviral activity against HIV at a low nanomolar concentration by interacting with high-mannose oligosaccharides on the virus surface envelope glycoprotein gp120. Atomic structures of wild-type CV-N revealed a monomer in solution and a domain-swapped dimer in the crystal, with the monomer comprising two independent carbohydrate binding sites that individually bind with micromolar affinity to di- and trimannoses. In the mutant CVN(mutDB), the binding site on domain B was abolished and the protein was found to be completely inactive against HIV. We determined the solution NMR and crystal structures of this variant and characterized its sugar binding properties. In solution and the crystal, CVN(mutDB) is a monomer and no domain-swapping was observed. The protein binds to Man-3 and Man-9 with similar dissociation constants ( approximately 4 muM). This confirms that the nanomolar activity of wild-type CV-N is related to the multisite nature of the protein carbohydrate interaction.

[1]  Alexander Wlodawer,et al.  The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of X-ray and NMR structures. , 2002, Structure.

[2]  S. Grzesiek,et al.  A simple and sensitive experiment for measurement of JCC couplings between backbone carbonyl and methyl carbons in isotopically enriched proteins , 1993, Journal of Biomolecular NMR.

[3]  J. Pflugrath,et al.  The finer things in X-ray diffraction data collection. , 1999, Acta crystallographica. Section D, Biological crystallography.

[4]  J. Thornton,et al.  AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR , 1996, Journal of biomolecular NMR.

[5]  P. V. van Zijl,et al.  Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation. , 1995, Journal of magnetic resonance. Series B.

[6]  R. Schnaar,et al.  High Affinity Binding of the Entamoeba histolytica Lectin to Polyvalent N-Acetylgalactosaminides (*) , 1995, The Journal of Biological Chemistry.

[7]  M. Newcomer,et al.  Protein folding and three-dimensional domain swapping: astrained relationship? , 2002 .

[8]  A. Gronenborn,et al.  The highly specific carbohydrate-binding protein cyanovirin-N: structure, anti-HIV/Ebola activity and possibilities for therapy. , 2005, Mini reviews in medicinal chemistry.

[9]  C. Bewley,et al.  Site-specific discrimination by cyanovirin-N for α-linked trisaccharides comprising the three arms of Man8 and Man9 , 2002 .

[10]  G. Wagner,et al.  An optimized 3D NOESY-HSQC. , 1996, Journal of magnetic resonance. Series B.

[11]  A. Bax,et al.  Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. , 1998, Journal of magnetic resonance.

[12]  H. Kunz,et al.  Multiple Sialyl Lewisx N‐Glycopeptides: Effective Ligands for E‐Selectin , 1996 .

[13]  K. Wüthrich,et al.  Torsion angle dynamics for NMR structure calculation with the new program DYANA. , 1997, Journal of molecular biology.

[14]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[15]  N. Guex,et al.  SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling , 1997, Electrophoresis.

[16]  Carole A Bewley,et al.  Potent inhibition of HIV-1 fusion by cyanovirin-N requires only a single high affinity carbohydrate binding site: characterization of low affinity carbohydrate binding site knockout mutants. , 2002, Journal of molecular biology.

[17]  Alexander Wlodawer,et al.  Structures of the Complexes of a Potent Anti-HIV Protein Cyanovirin-N and High Mannose Oligosaccharides* , 2002, The Journal of Biological Chemistry.

[18]  C. Sander,et al.  Errors in protein structures , 1996, Nature.

[19]  L Regan,et al.  An inverse correlation between loop length and stability in a four-helix-bundle protein. , 1997, Folding & design.

[20]  G M Whitesides,et al.  Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems. , 1994, Journal of medicinal chemistry.

[21]  G. Whitesides,et al.  Polyacrylamides Bearing Pendant α-Sialoside Groups Strongly Inhibit Agglutination of Erythrocytes by Influenza Virus: The Strong Inhibition Reflects Enhanced Binding through Cooperative Polyvalent Interactions , 1996 .

[22]  G. N. Ramachandran,et al.  Conformation of polypeptides and proteins. , 1968, Advances in protein chemistry.

[23]  T. Logan,et al.  A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments , 1993, Journal of biomolecular NMR.

[24]  Ad Bax,et al.  Methodological advances in protein NMR , 1993 .

[25]  A. Gronenborn,et al.  Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition. , 2006, Protein engineering, design & selection : PEDS.

[26]  Bruce A Johnson,et al.  Using NMRView to visualize and analyze the NMR spectra of macromolecules. , 2004, Methods in molecular biology.

[27]  C. Bewley,et al.  The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120. , 2001, Journal of the American Chemical Society.

[28]  R. Roy,et al.  Polysialosides scaffolded on p-Tert-butylcalix[4]arene , 1996 .

[29]  David Eisenberg,et al.  3D domain swapping: As domains continue to swap , 2002, Protein science : a publication of the Protein Society.

[30]  A. Gronenborn,et al.  Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping. , 1999, Journal of molecular biology.

[31]  Richard E. Moore,et al.  ANTIVIRAL ACTIVITY OF CULTURED BLUE‐GREEN ALGAE (CYANOPHYTA) 1 , 1993 .

[32]  Luke S. S. Guo,et al.  Sialyl Lewis x Liposomes as a Multivalent Ligand and Inhibitor of E-Selectin Mediated Cellular Adhesion , 1996 .

[33]  M. Boyd,et al.  Domain-swapped structure of a mutant of cyanovirin-N. , 2002, Biochemical and biophysical research communications.

[34]  A. Gronenborn,et al.  Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization. , 2002, Chemistry & biology.

[35]  L K Pannell,et al.  Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development , 1997, Antimicrobial agents and chemotherapy.

[36]  A. Gronenborn,et al.  The domain-swapped dimer of cyanovirin-N contains two sets of oligosaccharide binding sites in solution. , 2002, Biochemical and biophysical research communications.

[37]  M. Boyd,et al.  Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-specific manner. , 2001, Molecular pharmacology.

[38]  D. Bundle,et al.  Carbohydrate-protein interactions in antibodies and lectins , 1992, Current Biology.

[39]  A. Gronenborn,et al.  Solution structure of cyanovirin-N, a potent HIV-inactivating protein , 1998, Nature Structural Biology.

[40]  J. Paulson,et al.  Conformational analysis of sialyloligosaccharides. , 1991, Carbohydrate research.

[41]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[42]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Likelihood-enhanced Fast Translation Functions Biological Crystallography Likelihood-enhanced Fast Translation Functions , 2022 .

[43]  M. Boyd,et al.  Analysis of the interaction between the HIV-inactivating protein cyanovirin-N and soluble forms of the envelope glycoproteins gp120 and gp41. , 2000, Molecular pharmacology.

[44]  L. Kiessling,et al.  Recognition Specificity of Neoglycopolymers Prepared by Ring-Opening Metathesis Polymerization , 1996 .

[45]  Torsten Herrmann,et al.  Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. , 2002, Journal of molecular biology.

[46]  Y. Lee.  Biochemistry of carbohydrate‐protein interaction 1 , 1992, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[47]  Ad Bax,et al.  Validation of Protein Structure from Anisotropic Carbonyl Chemical Shifts in a Dilute Liquid Crystalline Phase , 1998 .

[48]  L. Kiessling,et al.  Strength in numbers: non-natural polyvalent carbohydrate derivatives. , 1996, Chemistry & biology.

[49]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.

[50]  Mike Carson,et al.  RIBBONS 2.0 , 1991 .

[51]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[52]  M. Boyd,et al.  Selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-N with high-mannose oligosaccharides on gp120 and other glycoproteins. , 2001, The Journal of pharmacology and experimental therapeutics.

[53]  Petra Fromme,et al.  A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity. , 2007, Biochemistry.

[54]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[55]  N. Sharon,et al.  Lectins: Carbohydrate-Specific Proteins That Mediate Cellular Recognition. , 1998, Chemical reviews.

[56]  A. Gronenborn,et al.  Atomic mapping of the interactions between the antiviral agent cyanovirin-N and oligomannosides by saturation-transfer difference NMR. , 2004, Biochemistry.

[57]  S. Fesik,et al.  Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules , 1990, Quarterly Reviews of Biophysics.

[58]  L. Kay,et al.  A gradient 13C NOESY-HSQC experiment for recording NOESY spectra of 13C-labeled proteins dissolved in H2O , 1993 .

[59]  Kanagaraj Sekar,et al.  RPMS: Ramachandran plot for multiple structures , 2008 .