|Director : Pedro M. Alzari (firstname.lastname@example.org)|
The research activities of the Unit of Structural Biochemistry are oriented towards the study of the three-dimensional structure and ligand-binding specificity of proteins by X-ray crystallography, protein biochemistry, microcalorimetry and molecular modelling techniques. The major themes of research involve structural studies of enzymes from microbial pathogens (mycobacteria, trypanosomatids), bacterial glycosidases and protein-carbohydrate complexes in different biological systems.
Bacterial glycosidases, carbohydrate recognition (B.G. Guimaraes, F. Schaeffer, H. Souchon, D. Tello, P.M. Alzari)
The bacterial cellulosome is a macromolecular agregate that is very efficient in degrading natural cellulose. The quaternary organization of the cellulosome depends on specific interactions between dockerin domains carried by the catalytic components of the complex and cohesin domains, which are individual receptor subunits linearly arranged within a non-catalytic scaffolding polypeptide. To gain further insights on the mechanism of cellulosomal assembly, we have determined the crystal structure of a single cohesin domain from the scaffolding protein and we are studying the cohesin-dockerin association by isothermal titration calorimetry spectroscopy. In collaboration with P. Béguin (IP), we have also attempted to structurally characterize the catalytic domains of cellulosomal glycosidases belonging to different protein families. Recently, we have determined the 3D structures of family 8 endoglucanase CelA and family 48 cellobiohydrolase CelS, both of which possess a similar alpha-barrel topology. The atomic (0.94 Å) resolution structure of CelA in complex with its saccharidic substrate showed that the substrate binds to an open acidic groove, where sugar distortion is found to play a direct role in catalysis. In contrast, the substrate-binding site in CelS is a long tunnel, in agreement with its exo function. However, despite their different substrate specificity and endo/exo modes of action, the structural comparison of CelA and CelS reveals a common catalytic center and mechanism of action, providing an interesting example of the evolutionary adaptation of a common structural motif to different functions.
Other structural studies of protein-sugar interactions in progress include the molecular recognition of the Tn determinant, a tumor-associated structure, by specific antibodies and plant lectins (in collaboration with E. Osinaga, Facultad de Medicina, Uruguay) and the immune recognition of the lipopolysaccharide from Vibrio cholerae (in collaboration with J.M. Fournier, IP, and P. Kovac, NIH, USA).
Trypanosomal enzymes (M.F. Amaya, A. Buschiazzo, P.M. Alzari)
The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans-sialidase (sialyltransferase) that is required to establish the infection in the mammalian host. Trypanosomes are unable to synthesize sialic acid and use this enzyme to scavenge the monosaccharide from host glycoconjugates to sialylate mucin-like acceptor molecules present in the parasite plasma membrane. In collaboration with the laboratory of A.C. Frasch (Univ. San Martin, Argentine), we are carrying out a structural and functional study of trypanosomal trans-sialidases from pathogenic (T. cruzi, T. brucei) and non-pathogenic (T. rangeli) parasites in order to understand their unusual enzymatic activities and to design specific inhibitors with potential therapeutic applications. We first determined the 3D structure of the, natural sialidase from T. rangeli at 2.2 Å resolution and showed that these enzymes fold into a catalytic b-propeller domain tightly associated with a lectin-like domain. We subsequently carried out structure-based exchange mutagenesis and enzyme kinetics studies of the T. cruzi and T. rangeli sialidases to identify amino acid residues which are critical for function. More recently, we have designed a surface mutant of the T. cruzi enzyme to facilitate crystallization and determined its 3D structure at 1.6 Å resolution. This structural framework provides a unique system to understand the molecular basis of sialyl-transfer activity in trypanosomes and, given the absence of a similar enzyme in eukaryotic cells, it provides an ideal target for the development of inhibitors useful in controlling the infection.
The capability of T. cruzi to adhere and invade host cells is aided by complex mechanisms allowing the parasite to evade the host immune response and establish a chronic infection. One of these mechanisms is the B/T polyclonal stimulation due to the presence of mitogenic/superantigenic molecules, leading to a non-specific immune response. Recently, the group of P. Minoprio at the IP identified a proline racemase as one such B-cell mitogenic factor in T. cruzi. The structural study of this trypanosomal racemase is currently in progress to elucidate its catalytic mechanism and to identify the putative regions of the protein that could be responsible for its mitogenic activity.
Mycobacterial enzymes involved in antibiotic resistance (B. Gomes Guimaraes, H. Souchon, P.M. Alzari)
The reemergence of tuberculosis in both developing and industrialized countries is a consequence of the appearance of M. tuberculosis strains resistant to many of the front-line compounds, including isoniazid (INH), that are currently utilized to treat the disease. The mechanism of action of INH is not completely understood but several lines of evidence indicate that the catalase-peroxidase KatG (which activates INH) and the alkyl hydroperoxidase AhpC (whose expression is up-regulated in some INH-resistant strains) are directly involved in the process. In collaboration with S.T. Cole (IP), we are currently carrying out a structural and functional study of the two mycobacterial proteins, KatG and AhpC, in order to gain further insight into the molecular mechanisms that account for isoniazid resistance in M. tuberculosis. During the last few months, we have determined the 3D structure of AhpC at 2.2 Å resolution and the crystallographic refinement is currently in progress.
Mycobacterial Ser/Thr protein kinases and phosphatases (B. Boitel, M. Ortiz, T. Nguyen, P.M. Alzari)
Bacterial signalling involve primarily the action of two-component systems, a histidine protein kinase and a response regulator. However, during the last few years several bacterial genes coding for eukariotic-like protein kinases or phosphatases have been identified. In particular, the genome of M. tuberculosis includes 11 genes which code for putative Ser/Thr protein kinases and 3 genes which code for putative Tyr or Ser/Thr protein phosphatases. We have started a systematic biochemical and structural study of mycobacterial Ser/Thr protein kinases and phosphatases to investigate the molecular basis of their modes of action and their possible role in cell signalling. Most enzymes have been subcloned in appropriate expression vectors and produced in bacteria in a soluble active form. Further biochemical and structural characterization of the recombinant proteins is in progress.
The apoptosis-inducing factor AIF and homologous flavoproteins from M. tuberculosis (M.J. Mate Perez, M. Ortiz, B. Boitel, D. Tello, P.M. Alzari)
Mitochondria play a key part in the regulation of apoptosis; their intermembrane space contains several proteins that are liberated through the outer membrane to participate in the degradation phase of apoptosis. Such apoptogenic proteins include cytochrome c, various pro-caspases, the IAP inhibitor Smac/DIABLO and a novel apoptosis-inducing factor, AIF. The latter is a caspase-independent death effector, which is able to translocate to the cell nucleus and induce chromatin condensation and large-scale DNA fragmentation. In collaboration with the laboratory of G. Kroemer (Villejuif, France), we have purified the AIF activity from mouse liver mitochondria and characterized its molecular properties. AIF is a flavoprotein with NADH oxidase activity, but its apoptogenic and enzymatic activities appear to be unrelated to each other. We are currently carrying out structural studies of AIF and homologous mycobacterial flavoproteins to investigate the structural basis of their enzymatic and apoptogenic mechanisms of action.
Biocalorimetry and structural thermodynamics (F. Schaeffer, P.M. Alzari)
The research activities in biocalorimetry aim to provide a quantitative description of the forces that govern the formation of biomolecular complexes. In combination with the structural analysis, this approach opens the way to the design of molecules with specific binding affinities based on thermodynamic principles. Methods are isothermal titration calorimetry, differential scanning calorimetry, with the development of statistical thermodynamics of molecular interactions which allows the deconvolution of the binding energetics in structural terms. Ongoing studies include the dockerin-cohesin interaction involved in bacterial cellulosome assembly (collaboration with P. Beguin, IP); protein tyrosine-kinase interaction and the design of new immunosuppressor drugs (collaboration with O.Acuto, IP); the interaction of human blood protein factors involved in coagulation and the design of new anti-coagulant drugs (collaboration with C. Bon, IP; C. Mounier, University of Cergy-Pontoise, M. Gelb, University of Washington); the interaction of transcription factors with promoter DNAs of genes involved in cancerisation and tumor cell invasion (collaboration with M. Aumercier & D. Sthéhelin, IP de Lille).
Cristallographic study of terminal desoxynucleotidyltransferase (T. Vatzakis, N. Expert-Bezancon, M. Delarue).
The structure of the catalytic domain of TdT has been solved at 2.35 Å resolution. A number of important biological implications of the structure have been found. In particular, it can be inferred from the structure that there is no open/close transition in this template-independent DNA polymerase. The structure of the binary complexes of TdT with an oligonucleotide primer and with an incoming dNTP have also been refined at 3.0 Å resolution. Different binary complexes with various dNTP and various divalent cations are being studied. This project will be continued with other structure-function studies of bacterial polymerases. The TdT project is a collaboration with J.B. Boule and C. Papanicolaou in F. Rougeon's lab (Unite de Génétique et de Biochimie du Développement).
Cristallographic study of M. tuberculosis TMP kinases (A. Haouz, M. Delarue)
The X-ray structure of this enzyme which is essential for nucleotide metabolism has been solved at 1.95 Å resolution in the presence of TMP. We use this structural information to design potent new inhibitors of this target. The enzyme is active in the crystal state. A new enzymatic and more direct dosage of the reaction has been devised using HPLC. The TMPK project is a collaboration with H. Munier-Lehmann and A.M. Gilles in O. Barzu's lab (Laboratoire de Chimie Structurale des Macromolécules).
Cristallisation de la protéine non-structurale NSP3 de rotavirus (N. Expert-Bezancon, M. Delarue)
The N-terminus part of NSP3 protein, which is responsible for the binding of the 3' end of viral mRNA, has been overexpressed and solubilized at suitable concentration for crystallization screens, in the presence of various oligoribonucleotides. This work is a collaboration with Rezorota: réseau de recherche sur les gastro-entérites à rotavirus (J. Cohen and D. Poncet, INRA, Jouy-en-Josas, France).
New refinement method and pahse combination protocol in crystallography (M. Delarue).
Mean Field theory has been used to recast in a single formalism the problem of phase optimization and phase combination, generalizing the approach of Blow and Crick (1959) and Sim (1960) to treat rigourously density modification techniques in the presence of an experimentally derived phase probability distribution function. The resulting combined maps are of much higher quality and should facilitate the use of automatic model building programs (Arp/wArp) in structural genomics projects.
|More informations on our web site|
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
FEJT, Françoise (email@example.com)
ALZARI, Pedro M. (Chef de laboratoire IP, firstname.lastname@example.org)
DELARUE, Marc (CR1 CNRS, email@example.com)
SCHAEFFER, Francis (Chargé de Recherche IP, firstname.lastname@example.org)
AMAYA, Maria Fernanda (PhD student, email@example.com)
BOITEL, Brigitte (Post-Doc, firstname.lastname@example.org)
BUSCHIAZZO, Alejandro (Post-Doc, email@example.com)
GOMES GUIMARAES, Beatriz (Post-Doc, firstname.lastname@example.org)
HAOUZ, Ahmed (Post-Doc, email@example.com)
MATE PEREZ, Maria Jesus (Post-Doc, firstname.lastname@example.org)
POMPEO, Frédérique (Post-Doc, email@example.com)
ORTIZ LOMBARDIA, Miguel (Post-Doc, firstname.lastname@example.org)
VATZAKI, Efstratia (Post-Doc, email@example.com)
EXPERT-BESANCON, Nicole (Ingénieur IP, firstname.lastname@example.org)
NGUYEN, Tong (Ingénieur IP, email@example.com)
SOUCHON, Hélène (ITA CNRS, firstname.lastname@example.org)
TELLO, Diana (Ingénieur IP, email@example.com)