Research project P5/33 (Research action P5)
The main goal of this IAP is to contribute to the elucidation of three major problems related to the structure of proteins and to the interactions they create with other proteins, nucleic acids or small molecules. Somewhat arbitrarily, this very general field has been divided in three major orientations which however significantly overlap : the folding of proteins, the structure-activity relationships and « complex systems » involving ligand-protein interactions and regulatory phenomena. Both experimental and theoretical approaches will be used. The chosen model systems have most often a direct impact on bacterial resistance to antibiotics, a topic of great practical importance.
1. Protein folding studies
The model proteins are lysozyme, active-site serine and Zn++-beta-lactamases and single chain antibody fragments from camelids (VHH) raised against these enzymes. Besides the analysis of the folding mechanisms of the individual proteins, the influence of the VHHs and, when applicable, of the metal ion, on the folding and unfolding of the corresponding protein will be studied. The structures of the enzyme-antibody complexes will be determined, allowing a detailed study of the protein-protein interaction zones. In parallel, a theoretical approach will be developed, based on the analysis of existing 3D structures and on the development of new algorithms for energy calculations.
2. Catalytic activity and structure-function relationships
The model enzymes recognize beta-lactam antibiotics, D-amino acids or D-amino acid-containing peptides or several of these structures. DD-peptidases, also called Penicillin-Binding Proteins (PBPs) are the targets of beta-lactam antibiotics in bacteria. They exhibit highly variable sensitivities to these compounds and the appearance of highly resistant enzymes explain the emergence of penicillin-resistant Enterococci and Staphylococci. At the present time, the structural factors responsible for the rates of reactions with penicillins remain poorly understood. Of related interest are the transglycosylases and D-alanine-D-alanine ligases which are potential new targets for antibacterial compounds. The first activity is often carried by the N-terminal domain of multimodular PBPs while the C-terminal domain catalyses the transpeptidation reaction. Our goals will be to obtain structural data on both domains, to try to understand the correlation between the two reactions and to discover inhibitors of the transglycosylase. Similarly, we will search for new inhibitors of the ligases. In turn, beta-lactamases are responsible for most resistance phenomena. We will continue our studies on their mechanisms of action and the design of new inhibitors. The last group of model enzymes are D-alanyl aminopeptidases which are not sensitive to penicillins despite the fact that they recognize D-Ala-D-Ala-terminated peptides. Structural analysis and the application of directed evolution techniques will shed light on the interactions responsible for the recognition of their specific substrates by the different enzymes. The catalytic pathways of all these enzymes will be studied by ab initio theoretical chemistry methods to obtain information about the structure of the stable and unstable intermediates, particularly about the transition states. The synthesis and evaluation of inhibitors will be performed on the basis of both experimental and theoretical approaches.
3. Complex systems : proteomic and biochemical approaches
If inactivation of the transpeptidases is the primary effect of penicillin in many bacteria, additional phenomena can occur such as autolysin activation and beta-lactamase induction. We plan to investigate the reactions of the bacterial cells in the presence of penicillin both by analysing the proteome and by searching for modified enzyme activities or metabolite concentrations. These two approaches should converge to a detailed explanation at the cellular level. Multimodular PBPs are also involved in bacterial cell division, together with a vast number of other proteins which form a supra-molecular assembly called the divisome. The structure and functioning of the divisome will be explored. New algorithms will be developed for better prediction of the protein-protein-interaction sites. Finally, bacterial pathogenicity often rests on the formation of biofilms. It will be attempted to advance our understanding of biofilm initiation and development in order to control and prevent their formation.
