Unit: Structural Biochemistry
Director: ALZARI, Pedro M.
Our research activities 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 modeling techniques. Current research subjects in the lab include the structural and functional studies of different enzymes (protein kinases and phosphatases) involved in bacterial cell signaling, glycosidases and glycosyltransferases of biomedical relevance, and hypothetical mycobacterial proteins of unknown function that could represent new potential therapeutic targets for drug design. The principal research subjects are described below; further information on these and other topics 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 ; Amaya et al, Structure, 2004). The 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 have been extended to other mycobacterial kinases such as PknD, PknE and PknF, all of which are also autophosphorylated in their respective activation loops (Duran et al, submitted). 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, the European Union 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). At present, more than 300 mycobacterial genes have been subcloned into bacterial expression vectors, several purified proteins have been subjected to crystallization trials and several crystal structures have been determined at high resolution. Some of these correspond to mycobacterial genes of unknown function, for which the structural information has provided useful hints to elucidate their putative biochemical function. The up-to-date status of the project is described in the dedicated Web site "Structural Genomics of Mycobacteria" (http://www.pasteur.fr/SGM).
Experimental and computational molecular structural biology (M. Delarue)
A. Crystal structures and protein crystallography
1. 6PGL (coll. PT6 and V. Stoven, Bioinformatique Structurale, I.P.)
The structure of 6-phosphogluconolactonase (6PGL) of T. brucei has been solved at 2.1 Angstrom resolution using one heavy-atom derivative with anomalous contribution and solvent flattening (SIRAS). The chain tracing was greatly facilitated by diffraction data collected on SeMet-protein crystals. The structure is now fully refined and finished, opening the way for structure-inspired drug design using 6PGL as a target.
2. Tdt and Pol mu (N. Expert-Bezancon, coll. F. Rougeon, I.P.)
The refinement of various complexes of Tdt has been completed and is being written up. Numerous purifications and crystallisation experiments have been done on polymerase mu, in an attempt to solve the structure of this important enzyme closely related to TdT and involved in Non-Homologous End Joining. The help of the Plateau Technique PT5 and PT6 is greatly appreciated.
B. Normal Modes and Structural Biology (E. Lindahl)
The work on Normal Modes has been continued in different directions, including their applications in X-Ray refinement, EM refinement, docking of small molecules, and structural alignment.
The first part has been published in 2004 while the second part has just been submitted for publication. In addition, a collaboration with the group of J.P. Changeux (I.P.) on the application of NMA to the nicotinic receptor (a pentameric protein) using the Elastic Network Model has been submitted and is currently under review. A Web site has been developed (http://lorentz.immstr.pasteur.fr) where NMA refinement can be performed on line and where various tools of modeling have been put on-line as well.
C. Solvent Structure and electrostatics (C. Azuara)
A new model of protein-solvent interactions has been developed, as a collaboration with H. Orland (CEA). It allows to get rid of the constant dielectric medium hypothesis and directly computes the solvent density around the solute. A program has been written to solve the equations, using the multi-grid method, making it a useful tool for computationals biologists. This will also be put on line in the future. The following issues are currently being (computationally) addressed:
- Incorporation of the protein polarisability using Normal Mode Analysis
- Study denaturation of proteins by urea as a function of concentration
- Study of stability of proteins in organic solvents (and mixtures thereof).
- Applications to protein crystallography and periodic systems: link with experimental data (electron density of the solvent)
Keywords: structural biology, X-ray diffraction, bacterial protein kinases, glycosidases and glycosyl transferases, tuberculosis, Trypanosoma cruzi, polymerases, structural genomics