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  Director : ALZARI, Pedro M. (alzari@pasteur.fr)



The apoptosis-inducing factor AIF (P.M. Alzari)

Mitochondria play a key part in the regulation of apoptosis; their intermembrane space contains several proteins that are released 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 the apoptosis-inducing factor, AIF. The latter is a flavoprotein that can stimulate a caspase-independent cell-death pathway required for early embryonic morphogenesis. 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. During the last year, we have determined the crystal structure of mouse AIF at 2.0 Å. Its active site structure and redox properties suggest that AIF functions as an electron transferase with a mechanism similar to that of bacterial ferredoxin reductases, its closest evolutionary homologs. However, AIF structurally differs from these proteins in some essential features, including a long insertion in a C-terminal eta-hairpin loop, that might be related to its emerging apoptogenic functions (Mate et al, Nature Struct. Biol., 2002).



Trypanosomal enzymes (P.M. Alzari)

Trans-sialidases (TS) are GPI-anchored surface enzymes expressed in specific developmental stages of trypanosome parasites like Trypanosoma cruzi, the etiologic agent of Chagas disease, and T. brucei, the agent of sleeping sickness. 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, a reaction that is critical for T. cruzi survival and cell invasion capability. In collaboration with A.C. Frasch (Argentine) and S. Withers (Canada), we are carrying out structural and biochemical studies 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 provide a framework for the structure-based design of specific inhibitors with potential therapeutic applications.

During the last year, we have determined the structures of the recombinant T. rangeli sialidase, alone and in complex with sialidase inhibitors and substrates, at 1.6 Å resolution. The protein exhibits a topologically rigid active site architecture that is unaffected by ligand binding. The overall binding of an inhibitor (DANA) to the active site cleft is similar to that observed in other viral and bacterial sialidases, dominated by the interactions of the inhibitor carboxylate with the conserved arginine triad. However, the interactions of the other pyranoside ring substituents (hydroxyl, N-acetyl and glycerol moieties) differ between trypanosomal, bacterial and viral sialidases, providing a structural basis for specific inhibitor design (Amaya et al, J. Mol. Biol., 2003).

We also determined the crystal structure of the trans-sialidase from T. cruzi at 1.6 Å resolution using a systematic surface-mutagenesis approach. The structures of several enzyme-sugar complexes demonstrate that, in contrast with most sialidases, sialic acid-binding triggers a conformational switch in trans-sialidase, which modulates the affinity for the acceptor substrate and concomitantly creates the conditions for efficient transglycosylation. A detailed comparison with the closely related structure of T. rangeli sialidase reveals a highly conserved catalytic center, where subtle structural differences account for strikingly different enzymatic activities and inhibition properties (Buschiazzo et al, Mol. Cell, 2002).

In parallel with these studies, we were able to identify and characterize the gene encoding the trans-sialidase from the African trypanosome T. brucei,. As in T. cruzi, the T. brucei enzyme displays both hydrolase and transferase activities. Site-directed mutagenesis of key functional residues revealed that the T. brucei enzyme has substrate specificity and catalytic properties similar to that of the T. cruzi enzyme (Montagna et al, Eur. J. Biochem., 2002). Crystals of the T. brucei trans-sialidase have been obtained and its structural characterization is currently in progress.

Mycobacterial Ser/Thr protein kinases and phosphatases (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. In collaboration with S.T. Cole's team at the IP, we are carrying out a 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. We have previously cloned several of these enzymes in appropriate expression vectors and produced the recombinant proteins for further biochemical and structural characterization.

During the last year, we have focused our work on two of these enzymes, the Ser/Thr protein kinase PknB and the phosphoSer/Thr phosphatase PstP. The corresponding genes pknB and pstP are found within an operon that is highly conserved in actinobacteria and could be involved in the regulation of peptidoglycan synthesis during bacterial cell growth. Both Pknb and PstP were expressed in E. coli then purified to homogeneity for biochemical characterization (work in progress). We have also crystallized and determined the 3D structure of PknB, confirming the conservation of the protein kinase fold across the evolutionary distance between high eukaryotes and eubacteria. The structure of PknB in complex with an ATP analog reveals the enzyme in its active state. However, the activation loop is partially disordered, suggesting an " induced fit " mode of binding for the so far unknown substrates of this kinase. Further work aiming at the identification of its physiological substrates and the structural characterization of kinase-inhibitor complexes is currently underway.

La génomique structurale des mycobactéries (P.M. Alzari)

The availability of massive amounts of data from genomic projects is imposing a novel dynamics to structural biology, in particular promoting the introduction of high-throughput methodologies into the discipline. This is possible thanks to the remarkable technological advances in a number of disciplines such as gene cloning, protein expression and purification, structure determination methods, crystallogenesis, synchrotron radiation sources, computing science and cryocrystallography. We wish to use these technologies to contribute towards the development of a better chimiotherapy for tuberculosis. In particular, we propose a structural genomics approach as a tool for the discovery of novel drug targets in mycobacteria.

Supported by grants from the National Genopole Programme, two European projects (X-TB and SPINE) and the Institut Pasteur, we have undertaken a structural genomics project on mycobacterial proteins, in collaboration with several other laboratories within the Institute. The initial list of targets includes a number of proteins that are known to be important for mycobacterial survival and/or virulence, as well as many others of unknown biological function but whose relevance as potential therapeutic targets is highlighted by the comparative analysis of mycobacterial genomes (M. tuberculosis, M. leprae). During the last year, we have undertaken the installation of the technological platforms required for this project and started the systematic cloning of mycobacterial genes, followed by protein production and crystallization trials. At present, over one hundred genes have been cloned in bacterial expression vectors and the first purified proteins are currently being subjected to crystallization assays.

Biocalorimetry and structural thermodynamics (F. Schaeffer)

The research activities in biocalorimetry aim at providing 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 collaborative studies include the characterization of dockerin-cohesin interactions in bacterial cellulosomal assembly (Schaeffer et al, Biochem., 2002; Miras et al, Biochem., 2002), the study of the ligand-binding properties of YajQ, an E. coli protein of unknown function (Saveanu et al, Prot. Sci., 2002), and the study of protein-protein interactions between human blood factors (in collaboration with C. Bon, IP) or between protein Tyr-kinases involved in T cell signalling (in collaboration avec O. Acuto, IP).

Cristallographic study of terminal desoxynucleotidyltransferase (M. Delarue).

The TdT project is a collaboration with with F. Rougeon's lab (Unité de Génétique et de Biochimie du Developpement, IP). The structure of the catalytic domain of TdT was solved at 2.35 Å resolution in 2001 (Delarue et al, EMBO J., 2002). 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 dA5 has been refined to a much better resolution than before (2.1 Angstrom resolution) and will be described in a future publication. Different binary complexes with various dNTP and various divalent cations have been solved and will be described in full detail when the refinement is complete. This project will be continued with other structure-function studies of bacterial polymerases.

In addition, a simplified version of the normal mode analysis of biological macromolecules has been used to investigate, in all cases where it is possible, the transition between the open and the closed forms of polymerases. It turns out that a handful of the lowest frequency normal modes calculated from the open form structure are sufficient to explain most of this transition, irrespective of the structural class to which this polymerase belong (Delarue et al, J. Mol. Biol., 2002). This very general result will be exploited to generate plausible intermediate structures between the different forms, to better understand the molecular nature of the transition.

Cristallographic study of M. tuberculosis TMP kinases (M. Delarue)

The X-ray structure of this enzyme which is essential for nucleotide metabolism was solved earlier at 1.95 Å resolution in the presence of TMP. We use this structural information to design potent new inhibitors of this target. We now have a better understanding of the specificity of the active site of the M. tuberculosis enzyme, compared to the human enzyme, especially concerning the TMP-bound Mg++ ion observed in the M. tuberculosis enzyme. Two new structures of complexes have been solved: one is a complex with an analogue of TMP, 5-CH2OH-dUMP, and the other is a complex with the bisubstrate inhibitor Ap5T. In the first case, the structure lead us to propose a mechanism based on a "classical" catalytic triad made of strictly conserved residues which had previously escaped attention. It is functioning in the reverse sense, namely by providing a proton donor to reprotonate the transferred phosphoryl group. This is the first time such a mechanism is described in detail for an NMP kinase. In the second case, we observed an unusual and unexpected binding site for the ATP moiety of Ap5T, compared to what is known in both the E. coli and yeast enzymes. This lead us to the proposal of a new family of inhibitors, specific of M. tuberculosis (Haouz et al, J. Biol. Chem., 2002).

Keywords: structural biology, bacterial protein kinases, glycosidases and glycosyl transferases, tuberculosis, Trypanosoma cruzi, structural genomics

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  Office staff Researchers Scientific trainees Other personnel
  Fejt, Françoise (ffejt@pasteur.fr) Alzari, Pedro M., IP (alzari@pasteur.fr)

Delarue, Marc, CNRS (delarue@pasteur.fr)

Schaeffer, Francis, IP (fschaeff@pasteur.fr)

Amaya, Maria Fernanda, Post-doc (amaya@pasteur.fr)

Azuara, Cyril, PhD student (azuara@pasteur.fr)

Buschiazzo, Alejandro, Post-doc (alebus@pasteur.fr)

Guerin, Marcelo, Post-doc (mguerin@pasteur.fr)

Fernandez, Pablo, Post-doc (fpablo@pasteur.fr)

Hagnerelle, Xavier, PhD student (hagnerel@pasteur.fr)

Villarino, Andrea, Post-doc (avilarin@pasteur.fr)

Wehenkel, Annemarie, PhD student (wehenkel@pasteur.fr)

Bellune, Alban, IP

Expert-Besançon, Nicole, IP (nexpert@pasteur.fr )

Formentin, Chantal, IP

Nguyen, Tong, IP (tong@pasteur.fr )

Souchon, Hélène, CNRS (hsouchon@pasteur.fr )

Tello-Manigne, Diana, IP (dtello@pasteur.fr )

Toscan, Isabelle, IP

Activity Reports 2002 - Institut Pasteur

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