Unit: Structural Biochemistry
Director: ALZARI, Pedro
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. During the last year, several new crystal structures of proteins and protein complexes have been determined in the laboratory. Major results include the structural studies of trypanosomal trans-sialidases, bacterial glycogen synthases, the proline racemase from Trypanosoma cruzi and several proteins from Mycobacterium tuberculosis that represent putative therapeutic targets. The main lines of research in the laboratory are described below; further information on these and other research subjects can be found in our Web page http://www.pasteur.fr/recherche/unites/Bstruct.
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.
We have previously determined the crystal structures of the recombinant T. rangeli sialidase (Buschiazzo et al, EMBO J, 2000) and T. cruzi trans-sialidase (Buschiazzo et al, Mol. Cell, 2002), and carried out mutagenesis studies of the enzymes from T. cruzi (Paris et al, Glycobiol., 2001) and T. brucei (Montagna et al, Eur. J. Biochem., 2002). These studies have shown that, in contrast with sialidases, the active site architecture of trans-sialidases is intrinsically flexible. Binding of sialylated substrates triggers a conformational switch, 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. In addition, the overall mode of binding of a transition state-analog inhibitor to the active site cleft of trans-sialidase 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).
Research efforts during the last few years have substantiated the pathogenic role of microbial sialidases in a number of infectious diseases, making of these enzymes attractive targets for drug design. Besides trypanosomes, a prominent example is influenza virus infection, for which structural biology and rational drug design have led to the development of at least three potent sialidase inhibitors clinically useful as anti-viral compounds. However, in spite of extensive studies on the structure and mechanism of sialidases, many fundamental questions on their catalytic mechanism still remain unanswered or controversial, in large part because no structural information is currently available on the different enzymatic species along the reaction coordinate. The T. cruzi trans-sialidase provides an ideal system to address this issue, since the higher anticipated affinities in the aglycone site improve the chances of observing bound or intermediate species. Thus, using an activated (fluor-containing) sialoside substrate, we were able to trap the reaction intermediate and show that trans-sialidase (and likely all microbial sialidases) operates through a double displacement mechanism, involving the transient formation of a covalent sialyl-enzyme intermediate with a strictly conserved tyrosine residue (Watts et al, J. Am. Chem. Soc, 2003). We also determined the atomic structures of this covalent intermediate and those of two competent enzyme-substrate (Michaelis) complexes at 1.6 Å resolution by X-ray diffraction techniques (Amaya et al, 2003, submitted). These snapshots along the catalytic cycle demonstrate that catalysis by sialidases and trans-sialidases occurs via a similar mechanism to that of other retaining glycosidases, but with some intriguing differences that may have evolved in response to the substrate structure.
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.
More recently, we have focused our work on the catalytic domains of two trans-membrane enzymes from Mycobacterium tuberculosis, the Ser/Thr protein kinase PknB and the protein phosphatase PstP, which are part of a conserved operon presumably involved in cell growth control. PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn2+-dependent manner. Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation. Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase (Ortiz et al, J. Biol. Chem., 2003), namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation. Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role. These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth (Boitel et al, Mol. Microbiol. 2003). The phosphorylation studies are currently being extended to other mycobacterial kinases such as PknD, PknE and PknF, all of which also appear to be autophosphorylated in their respective activation loops. Further work aiming at the identification of physiological substrates and the structural characterization of kinase-inhibitor complexes is currently underway.
Structural genomics of mycobacteria (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 Network, two European projects (X-TB and SPINE) and the Institut Pasteur, we have undertaken a structural genomics project on mycobacterial proteins, in collaboration with 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). Two technical platforms have been setup for protein production (http://www.pasteur.fr/recherche/genopole/PF5) and high-throughput cloning and protein crystallization (http://www.pasteur.fr/recherche/genopole/PF6). The project is now well underway: more than 300 mycobacterial genes have been subcloned into bacterial expression vectors, several purified proteins have been subjected to crystallization trials and the first crystal structures have been determined during the last year. The up-to-date status of the project is described in the dedicated Web site "Structural Genomics of Mycobacteria" (http://www.pasteur.fr/SGM).
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. 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 Å 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.
We are also now studying structure-function relationship studies on human pol mu, a close "cousin" of TdT involved in NHEJ recombination process. 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. 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 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.
Keywords: structural biology, X-ray diffraction, bacterial protein kinases, glycosidases and glycosyl transferases, tuberculosis, Trypanosoma cruzi, polymerases, structural genomics"