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Institutes/ Institute of Biology and Technology Saclay (iBiTec-S)/ Units/ Molecular engineering of proteins (SIMOPRO)/ Molecular toxinology and biotechnology laboratory (LTMB)/ Crystallogenesis (E. Stura)

Crystallogenesis

Enrico STURA
CEA Saclay/Bât. 152


enrico.stura@cea.fr


iBiTec-S / SIMOPRO / LTMB


Moyens humains
Enrico STURA, Group Leader
Laura VERA, Research Technician


Thèmes de recherche

 
 
The main research themeof the laboratory is the crystallization of macromolecules.
 



©CEA/E. Stura

 
The goal is the development of new approaches while constantly improving the current techniques. Among the new developments is the use of antibodies and engineered bacterial proteins that bind immunoglobulins to induce crystallization of antigen-antibody complexes. We are studying the use of anti-GST and anti-Tag proteins to be used in the construction of protein scaffolds where the fusion protein and peptide tag is part of the target protein to be crystallized. This approach avoids having to produce new antibodies for each new target.
 



 ©CEA/E. Stura

 
The focus on complexes is because complexes between proteins, proteins and nucleic acids and proteins and peptides are biologically more interesting than the protein alone, but also because in such system there is greater scope for a combinatiorial approach that increases substantially the chances of crystallizing a particular protein in one complex or another, and although each complex is more difficult to crystallize, when the whole combinatorial ensamble is considered together, the overall chances of success increases, especially when the combinatorial system includes several partners. For example, when two antibodies directed towards the same antigen have distinct epitopes, they may be used in a sandwich complex.
 



©CEA/E. Stura

 
Among the work directed towards the improvement of current crystallization methods a substantial effort is dedicated to improving the efficiency screening techniques during the search for crystallization conditions. This is very important when only small amounts of protein are available. Once crystals are obtained, additives are used to improve the size and the diffraction quality of the resulting crystals. Additives are very important for simple tasks such as the control oxidation or reduction of free cysteines but also to modify nucleation and crystal growth. Our expertise in crystallogenesis is available to collaborators whose projects require structural data.


 
Protein engineering and immunology
IgG-derived Fc fragments
Among our collaborators, Dr. Dr. Sylvie Jorieux of the Unité de Recherche du LFB shares with us a particular interest in understanding the Fc portion of human immunoglobulins and how its structural variations affect its binding to the various Fc-gamma receptors. This fragment is also the target of a pathological response by the immune system, in particular rheumatoid factors.
 


©CEA/E. Stura

 
The Fc fragment is often used as a fusion partner for the expression of glycoproteins. We are attempting to obtain crystals of glycoproteins fused to Fc using rheumatoid factor Fabs.
 
 
Antibodies and antibody epitopes
Protein-protein interaction is a general focus of our research, but more specifically we are dedicated to antibody-antigen interactions where the antigen can be a cancer marker, a steroid, a peptide or a protein from a virus or pathogen. The Mixte BioMérieux-CEA under the direction of Dr. Frédéric Ducancel shares such interests and adds complementary expertize in antibody engineering in a mutually advantageous collaboration.
 
Nuclear and environmental toxicology
In collaboration with Eric Quemeneur, we have solved the crystal structures of two antibodies that recognize uranyl complexed to phenanthroline.
 
Toxins
We continue to collaborate with Denis Servent to understand the mode of interaction of the MT7 muscarinic toxin with the hM1 receptor. In this collaboration we have determined the crystal structures of several constructs of this toxin to understand the specific role of each loop. 
 
We are also interested in bacterial toxins that modify small G proteins such as Exoenzyme C3 from Clostridium botulinum that directs ADP-ribosylation of RhoA as well as the large clostridial toxins that target the same human protein.
 




Publications

Tlatli R, Nozach H, Collet G, Beau F, Vera L, Stura E, Dive V, Cuniasse P. (2013). Grafting of functional motifs onto protein scaffolds identified by PDB screening - an efficient route to design optimizable protein binders. FEBS J., 280, 139-159.

Siberil S, Menez R, Jorieux S, de Romeuf C, Bourel D, Fridman W H, Ducancel F, Stura E A, Teillaud J L. (2012). Effect of zinc on human IgG1 and its Fe gamma R interactions. Immunol Lett. 143, 60-69.

Stura EA, Le Roux L, Guitot K, Garcia S, Bregant S, Beau F, Vera L, Collet G, Ptchelkine D, Bakirci H, Dive V. (2012). Structural framework for covalent inhibition of Clostridium botulinum neurotoxin A by targeting Cys165. J Biol Chem. 287, 33607-33614.

Cockburn, J.J.B., Sanchez, M.E.N., Goncalvez, A.P., Zaitseva, E., Stura, E.A, Kikuti, C.M., Duquerroy, S., Dussart, P., Chernomordik, L.V., Lai, C.-J. & Rey, F.A. (2011) Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus EMBO J. 31, 767-79.

Igonet, S., Vaney, M.C., Vonhrein, C., Bricogne, G., Stura, E.A., Hengartner, H., Eschli, B. & Rey, F.A. (2011) X-ray structure of the arenavirus glycoprotein GP2 in its postfusion hairpin conformation. Proc. Natl. Acad. Sci. U.S.A. 108, 19967-72.

Muller, B.H., Savatier, A., L'hostis, G., Costa, N., Bossus, M., Michel, S., Ott, C., Becquart, L., Ruffion, A., Stura, E.A. & Ducancel, F. (2011) In Vitro Affinity Maturation of an Anti-PSA Antibody for Prostate Cancer Diagnostic Assay. J. Mol. Biol. 414: 545-562.

Stura, E.A., Muller, B.H., Michel, S., Jolivet-Reynaud, C. & Ducancel, F. (2011). Crystal structure of human prostate-specific antigen (PSA) in a sandwich antibody complex. J. Mol. Biol. 414: 530-544.

Vera, L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11: 2755–2762.

Devel L, Czarny B, Beau F, Georgiadis D, Stura E, Dive V. (2010). Third generation of matrix metalloprotease inhibitors: Gain in selectivity by targeting the depth of the S(1)' cavity. Biochimie. 92, 1501-1508.

Devel L, Garcia S, Czarny B, Beau F, Lajeunesse E, Vera L, Georgiadis D, Stura E, Dive V. (2010). Insights from selective non-phosphinic inhibitors of MMP-12 tailored to fit with a S1 ' loop canonical conformation. J Biol Chem. 285, 35900-35909.

Gross G, Gallopin M, Vandame M, Couprie J, Stura E, Zinn-Justin S, Drevet P. (2010). Conformational exchange is critical for the productivity of an oxidative folding intermediate with buried free cysteines. J Mol Biol. 403, 299-312.

Chruszcz M, Chapman MD, Vailes LD, Stura EA, Saint-Remy JM, Minor W, Pomes A. (2009).Crystal Structures of Mite Allergens Der f 1 and Der p 1 Reveal Differences in Surface-Exposed Residues that May Influence Antibody Binding. J Mol Biol. 386, 520-530.

Pawlak J, Mackessy SP, Sixberry NM, Stura EA, Le Du MH, Menez R, Foo CS, Menez A, Nirthanan S, Kini RM. (2009). Irditoxin, a novel covalently linked heterodimeric three-finger toxin with high taxon-specific neurotoxicity. Faseb J. 23, 534-45.

Fruchart C, Mourier G, Marquer C, Stura E, Birdsall NJ, Servent D. (2008). Different interactions between MT7 toxin and the human muscarinic M1 receptor in its free and NMS-occupied states. Mol Pharmacol. 74, 1554-63.

Ménétrey J, Flatau G, Boquet P, Ménez A, Stura EA. (2008). Structural basis for the NAD-hydrolysis mechanism and the ARTT-loop plasticity of C3 exoenzymes. Protein Sci. 17, 878-86.

Ménez R, Michel S, Muller BH, Bossus M, Ducancel F, Jolivet-Reynaud C, Stura EA. (2008). Crystal structure of a ternary complex between human prostate-specific antigen, its substrate acyl-intermediate and an activating antibody. J Mol Biol. 376, 1021-33.

Duquerroy S, Stura EA, Bressanelli S, Fabiane SM, Vaney MC, Beale D, Hamon M, Casali P, Rey FA, Sutton BJ, Taussig MJ. (2007). Crystal Structure of a Human Autoimmune Complex between IgM Rheumatoid Factor RF61 and IgG1 Fc Reveals a Novel Epitope and Evidence for Affinity Maturation. J Mol Biol. 368, 1321-31.

Menetrey J, Perderiset M, Cicolari J, Houdusse A, Stura EA. (2007). Improving diffraction from 3 to 2 angstrom for a complex between a small GTPase and its effector by analysis of crystal contacts and use of reverse screening. Cryst Growth Des. 7, 2140-2146.

Ménez R, Michel S, Muller BH, Bossus M, Ducancel F, Jolivet-Reynaud C, Stura EA. (2007).Crystal structure of a ternary complex between human prostate-specific antigen, its substrate acyl-intermediate and an activating antibody. J Mol Biol. 376, 1021-33.

Siberil S, Dutertre CA, Boix C, Bonnin E, Menez R, Stura E, Jorieux S, Fridman WH, Teillaud JL. (2006). Molecular aspects of human FcgammaR interactions with IgG: functional and therapeutic consequences. Immunol Lett. 106, 111-118.

Pawlak J, Mackessy SP, Fry BG, Bhatia M, Mourier G, Fruchart-Gaillard C, Servent D, Menez R, Stura E, Menez A, Kini RM. (2006). Denmotoxin, a Three-finger Toxin from the Colubrid Snake Boiga dendrophila (Mangrove Catsnake) with Bird-specific Activity. J Biol Chem. 281, 29030-2941.

Le Maire A, Schiltz M, Stura EA, Pinon-Lataillade G, Couprie J, Gondry M, Angulo JF, Zinn-Justin S. (2006). A Tandem of SH3-like Domains Participates in RNA Binding in KIN17, a Human Protein Activated in Response to Genotoxics J Mol Biol. 364, 764-776.

Magis C, Gasparini D, Lecoq A, Le Du MH, Stura E, Charbonnier JB, Mourier G, Boulain JC, Pardo L, Caruana A, Joly A, Lefranc M, Masella M, Menez A, Cuniasse P. (2006). Structure-based secondary structure-independent approach to design protein ligands: application to the design of Kv1.2 potassium channel blockers. J Am Chem Soc. 128, 16190-16205.

Llinas P, Masella M, Stigbrand T, Menez A, Stura EA, Le Du MH. (2006). Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones. Protein Sci. 15, 1691-1700.

Charbonnier JB, Christova P, Shosheva A, Stura E, Le Du MH, Blouquit Y, Duchambon P, Miron S, Craescu CT. (2006). Crystallization and preliminary X-ray diffraction data of the complex between human centrin 2 and a peptide from the protein XPC. Acta Crystallogr F. 62, 649-651.

Menez R, Housden NG, Harrison S, Jolivet-Reynaud C, Gore MG, Stura EA. (2005). Different crystal packing in Fab-protein L semi-disordered peptide complex. Acta Crystallogr D. 61, 744-749.

Llinas P, Stura EA, Menez A, Kiss Z, Stigbrand T, Millan JL, Le Du MH. (2005). Structural studies of human placental alkaline phosphatase in complex with functional ligands. J Mol Biol. 350, 441-451.

Llinas P, Le Du MH, Gardsvoll H, Dano K, Ploug M, Gilquin B, Stura EA, Menez A. (2005). Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide.EMBO J. 24, 1655–1663.

Granata V, Housden NG, Harrison S, Jolivet-Reynaud C, Gore MG, Stura EA. (2005). Comparison of the crystallization and crystal packing of two Fab single-site mutant protein L complexes. Acta Crystallogr D. 61, 750-754.

Graille M, Stura EA, Bossus M, Muller BH, Letourneur O, Battail-Poirot N, Sibai G, Gauthier M, Rolland D, Le Du MH, Ducancel F. (2005). Crystal structure of the complex between the monomeric form of Toxoplasma gondii surface antigen 1 (SAG1) and a monoclonal antibody that mimics the human immune response. J Mol Biol. 354, 447-458.

Dubreuil O, Bossus M, Graille M, Bilous M, Savatier A, Jolivet M, Menez A, Stura E, Ducancel F. (2005). Fine tuning of the specificity of an anti-progesterone antibody by first and second sphere residue engineering. J Biol Chem. 280, 24880-24887.

Housden NG, Harrison S, Housden HR, Thomas KA, Beckingham JA, Roberts SE, Bottomley SP, Graille M, Stura E, Gore MG. (2004). Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains. J Biol Chem. 279, 9370-9378.

Bressanelli S, Stiasny K, Allison SL, Stura EA, Duquerroy S, Lescar J, Heinz FX, Rey FA. (2004). Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO J. 25, 728-738.

Menez R, Bossus M, Muller BH, Sibai G, Dalbon P, Ducancel F, Jolivet-Reynaud C, Stura EA. (2003). Crystal structure of a hydrophobic immunodominant antigenic site on hepatitis C virus core protein complexed to monoclonal antibody 19D9D6. J Immunol.170, 1917-1924.

Housden NG, Harrison S, Roberts SE, Beckingham JA, Graille M, Stura E, Gore MG. (2003). Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus. Biochem Soc Trans. 31, 716-718.