http://www-dsv.cea.fr/diep/diep_uk.htm

CE Saclay - 91190 Gif-sur-Yvette - France

Phone: +33 1 6908 7133
Fax: +33 1 6908 9137

Email: claudio.vita@cea.fr
Research interests
Engineering novel proteins exhibiting a predetermined structure and a specific function is an exciting prospect of protein engineering. However, owing to our limited understanding of the relations between protein structure and function, this still represents a rather challenging task. To overcome this impasse at least in part, we have developed novel structurally well-defined and functional small proteins by transferring a target active site on a specific region of a protein structure, acting as a scaffold. Small disulfide-rich proteins, like scorpion toxins, are particularly attractive as structural scaffolds because of their conformational stability, permissiveness of sequence mutations, and because their small size allows easy chemical synthesis, incorporation of unnatural probes and three-dimensional structure resolution by NMR.

Engineering of a metal binding site. By utilizing this strategy, we have been able to engineer a three-histidine metal binding site on the b-sheet face of the charybdotoxin scaffold. This site bound heavy metals with distinct specificity. Structure resolution by 1H-NMR revealed that, in spite of the numerous mutations introduced, the structural scaffold is preserved, the structure of the binding site is well-defined and exhibits a square planar geometry, thus differing from the target tetrahedrical site of carbonic anhydrase. This small (37 AA) metal-binding protein represents the lead for the engineering of more complex binding sites and catalytic centers.

Engineering of a curaremimetic mini-protein. A similar strategy of active site transfer to a structural scaffold was utilized in the engineering of a miniprotein that presented the core of a neurotoxin curaremimetic surface, binding to the acethylcholine receptor, on the structurally equivalent b-sheet of the charybdotoxin scaffold. This chimera bound the acetylcholine receptor with low but specific activity and was able to induce antibodies specifically neutralizing the toxic effects of the parent neurotoxin.

Engineering of a mini-CD4, inhibitor of HIV-1 infectivity. In a recent application of our engineering approach, we reproduced the core of the CD4 surface binding to the gp120 glycoprotein of HIV-1 envelope on the b-sheet of charybdotoxin. The synthesized chimeric miniprotein was able to competitively inhibit gp120-CD4 interaction and elicit antibodies specifically recognizing CD4. A similar transfer was performed on the smaller scyllatoxin scaffold. This new chimera (27 AA only) binds gp120 and inhibits HIV-1 infection, albeit at high (10-5 M) concentration. Structure resolution by 1H-NMR and alanine scanning approach demonstrated that the CD4 site was structurally and functionally reproduced on the mini-protein with striking precision. These analyses also suggested some mutations which, once incorporated into the miniprotein, increased its affinity for gp120 by 100-fold. The improved mini-CD4 is an effective inhibitor of HIV-1 infection and prevents infection of competent cell lines and PBL with both laboratory-adapted and primary isolates. This mini-CD4 construction is the basis for the development of unique tools to study the complex process of HIV entry into cells and the design of anti-HIV drugs.

Engineering of chemokine antagonists. We set up a fast and efficient protocol for the synthesis of RANTES and other chemokine derivatives by using Fmoc-chemistry and stepwise solid-phase method. Specifically labeled chemokine derivatives, obtained with this method, have been patented. We are presently mapping RANTES surface(s) involved in receptor and glycosaminoglycan binding by chemical synthesis of RANTES derivatives. Our final goal is the engineering of chemokine receptor antagonists useful as tools in the study of HIV-1 entry and as leads in the development of antiviral drugs.

Recent Publications.

* B. Pierret, H. Virelizier and C. Vita. Synthesis of a metal binding protein designed on the a/b scaffold of charybdotoxin. Int. J. Peptide Protein Res. 1995, 46, 471-476

* C. Vita, C. Roumestand, F. Toma and A. Ménez. Scorpion toxins as natural scaffolds for protein engineering. Proc. Natl. Acad. Sci. USA 1995, 92, 6404-6408

* P. Hanson, G. Martinez, G. Millhauser, F. Formaggio, M. Crisma, C. Toniolo and C. Vita. Distinguishing helix conformation in alanine-rich peptides using the unnatural amino acid TOAC and electron spin resonance. J. Am. Chem. Soc. 1996, 118, 271-272

* E. Drakopoulou, S. Zinn-Justin, M. Guenneugues, B. Gilquin, A. Ménez and C. Vita. Changing the structural context of a functional b-hairpin. Synthesis and characterisation of a chimera containing the curaremimetic loop of a snake toxin in the scorpion a/b scaffold. J. Biol. Chem. 1996, 271, 11979-11987

* S. Zinn-Justin, M. Guenneugues, E. Drakopoulou, B. Gilquin, C. Vita and A. Ménez. Transfer of a b-hairpin from the functional site of snake curaremimetic toxins to the a/b scaffold of scorpion toxins: three-dimensional solution structure of the chimeric protein. Biochemistry 1996, 35, 8535-8543

* M. Dauplais, A. Lecoq, J. Song, J. Cotton, N. Jamin, B. Gilquin, C. Roumestand, C. Vita, C.L.C. de Madeiros, E.G., Rowan, A.L. Harvey and A. Ménez. On the convergent evolution of animal toxins. Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures. J. Biol. Chem. 1997, 272, 4302-4309.

* J. Song, B. Gilquin, N. Jamin, M. Guenneugues, M. Dauplais, E. Drakopoulou, C. Vita and A. Ménez. NMR solution structure of a two disulfide derivative of charybdotoxin: structural evidence for conservation of scorpion toxin a/b motif and its hydrophobic side chain packing. Biochemistry 1997, 36, 3760-3766.

* C. Vita. Engineering novel proteins by transfer of active sites to natural scaffolds. Curr. Opin. Biotechnol. 1997, 8, 429-434

* E. Drakopoulou, J. Vizzavona, J. Neyton, V. Aniort, F. Bouet, H. Virelizier, A. Ménez and C. Vita. Consequence of the removal of evolutionary conserved disulfide bridges on the structure and function of charybdotoxin and evidence that particular cystine spacings govern specific disulfide bond formation. Biochemistry 1998, 37, 1292-1301.

* E. Drakopoulou, J., Vizzavona and C. Vita. Engineering a CD4 mimetic inhibiting the binding of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 to human lymphocyte CD4 by the transfer of a CD4 functional site to a small natural scaffold. Lett. Pep. Sci. 1998, 5, 241-245

* L. Ylisastigui, J. Vizzavona, E. Drakopoulou, P. Paindavoine, C.-F. Calvo, M. Parmentier, J.C. Gluckman, C. Vita and A. Benjouad. Synthetic full-length and truncated RANTES inhibit HIV-1 infection of primary macrophages. AIDS 1998, 12, 977-984

* C. Vita, J. Vizzavona, E. Drakopoulou, S. Zinn-Justin, B. Gilquin and A. Ménez. Novel miniproteins engineered by the transfer of active sites to small natural scaffolds. Biopolymers (Peptide Science) 1998, 47, 93-100

* B. Bodaghi, T.R. Jones, D. Zipeto, C. Vita, L. Sun, L. Laurent, F. Arenzana-Seisdedos, J.L. Virelizier and S. Michelson. Chemokine sequestration by viral chemoreceptors as a novel viral strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. J. Exp. Med. 1998, 188, 855-866

* L. Ylistagui, S. Amzazi, Y. Bakri, J. Vizzavona, C. Vita, J.C. Gluckman and A. Benjouad. Effect of RANTES on the infection of monocyte-derived primary macrophages by human immunodeficiency virus tyte 1 and type 2. Biomed. & Pharmacother. 1998, 52, 447-453

* P. Hanson, D.J. Anderson, G. Martinez, G. Millhauser, F. Formaggio, M. Crisma, C. Toniolo and C. Vita. Electro spin resonance and structural analysis of water soluble, alanine-rich peptides incorporating TOAC. Molecular Physics 1998, 95, 957-968

* J. Song, N. Jamin, B. Gilquin, C. Vita and A. Ménez. A gradual disruption of tight side-chain packing: 2D 1H-NMR characterization of acid-induced unfolding of CHABII. Nat. Struct. Biol. 1999, 6, 129-134