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 and NMR 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-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 that were recently carried out in the laboratory 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.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. We have undertaken structural studies of this trypanosomal racemase 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 combinatin with the structural analyis, 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).