Research project P6/19 (Research action P6)
Modern DNA sequencing methods give rise to an enormous amount of data about the genomes of an increasing number of organisms. By contrast, we are far from being able to integrate all these data in a detailed understanding of the functioning of living cells. This paradox is explained by the fact that our knowledge of the interactions between biological molecules has progressed much more slowly than the determination of genomic sequences. Proteins are the most important macromolecules for the functioning of the cells and molecular interactions are the central phenomena which can explain their behaviours: the tertiary structures of proteins and the dynamics of these structures depend mainly on intramolecular interactions while their quaternary structures and their associations with ligands of all sizes rest on intermolecular interactions. These ligands can be substrates, inhibitors, effectors, nucleic acids, lipid bilayers or other proteins. Unwanted alterations of these interactions can transform a normal process into an aberrant one, resulting in many types of pathologies. Alternatively, antimicrobial chemotherapy rests on a perturbation of the metabolic processes of the parasite. The present network is devoted to the most fundamental aspects of protein science in order to contribute to filling the gap mentioned above.
For a protein to fulfil its normal function, it must fold into a specific structure. An increasing number of human diseases are recognised to be associated with aberrant folding processes, some of which lead to the aggregation of proteins in the form of amyloid fibers or plaques. In the case of the normal fold, one might consider that the remarkable cooperativity of the structure favours intramolecular interactions. In contrast, reduction in global cooperativity (e.g. due to mutations) allows transitions to abnormal folds and results in the formation of intermolecular interactions, which ultimately lead to aggregation.
A first objective is thus to try to understand the mechanism of this transition and to identify factors which might affect it. If the folding mechanism of some small soluble proteins (with ususally less than 100 residues) is relatively well documented, the situation is very different for membrane-bound and larger molecules. The study of the behaviour and stability of some of these proteins and of the effects of potential ligands (cofactors, inhibitors, substrates or other proteins) will represent an important facet of our approach. A second and closely related facet will deal with protein dynamics. It is well accepted that the instability of a protein is somehow related to its flexibility but function requires flexibility. The functional behaviour of a protein thus results from a delicate balance between stability and flexibility. This is further examplified by unstructured domains in the native fold of some proteins which are thought to acquire a unique conformation through interaction with various ligands.
The aim of the second objective is the study of protein-ligand interactions. The ligands can be small molecules (substrates, inhibitors, effectors), nucleic acids, large structures such as parts of the cell envelope and/or other proteins. The chosen model systems are the proteins responsible for bacterial cell wall metabolism and resistance to ß-lactam antibiotics. This implies to continue the search for new antibacterial drugs whose targets would be the transglycosylases and the DD-transpeptidases involved in peptidoglycan biosynthesis, the study of ß-lactamases and of efflux transporters. This field requires a major effort in the design and modeling of new molecules and in the synthesis of original inhibitors for the enzymes and of difficult to obtain substrates or substrate analogues. Induction of the synthesis of ß-lactamases also involves DNA-protein and DNA-protein-ligand interactions as well as the transmission of messages by transmembrane proteins, which provides a clear link to the first objective. Moreover, DD-transpeptidases and transglycosylases interact with other proteins to create membrane-associated supramolecular assemblages involved in cell elongation and division, which introduces the third objective: protein-protein interactions, their formation and disappearance according to the modifications of the cell physiology. When concerned with the stability of supramolecular assemblages, this approach is related to the first objective and will also involve the determination of the structures of membrane proteins, which, despite some recent successes, still presents some major technical difficulties. Finally, protein-protein interactions also determine the formation of biofilms a phenomenon closely related to bacterial antibiotic resistance and pathogenesis and, as recent results suggest, to the metabolism of peptidoglycan.
As a whole, the project thus presents a gradation from the study of the behaviour of individual molecules and of intramolecular forces (an approach which is reinforced by the contribution of theoretical chemists) to that of stable or transient supramolecular networks.
This ambitious program thus requires the analysis of a diversity of model systems. These systems were carefully chosen on the basis of the past experience of the participating groups and an important part of the program is devoted to the phenomenon of the bacterial resistance to antibiotics, the study of which presents clear contributions to the three objectives. In addition, problems related to protein dynamics, including the formation of amyloid fibres represent a very fundamental approach with very important applications in the medical field.
To contribute to solving these problems in the most efficient way, a collaboration has been established between the best Belgian teams in protein science. These groups will contribute the best possible technologies in molecular and structural biology, bacteriology, biophysics, bioinformatics, protein and membrane chemistry enzymology, organic synthesis and theoretical chemistry. The contribution of the European partner (Cam-UK, Dobson) is particularly precious in the fields of biophysics, structural biology and protein chemistry. The crystallographers have a privileged access to the ESRF facilities in Grenoble. Whenever possible or useful, exchange of technologies will take place between the participating laboratories, which will be especially interesting for the training of the graduate students.
