Molecular mechanisms of signaling in fatty acid synthesis of Gram-positive bacteria – ReguLipids+

1-Scientific background and objectives : The molecular understanding of lipid biosynthesis regulation in Gram+ bacteria is still fragmentary. To unravel the mechanisms of signal integration in two key pathways of lipid homeostasis in Bacillus subtilis, we will focus on the study of two proteins that play a central role, DesK and FapR. The synthesis of unsaturated fatty acids is tightly controlled in response to cold shock, through a two-components system. This system includes a trans-membrane histidine-kinase, DesK, from which we have recently solved the 3D structure of the cytoplasmic domain. DesK detects a drop in environmental temperature, autophosphorylates and transfers thereafter the phosphate to its response regulator partner DesR, which finally regulates the expression of a key desaturase. We wish to understand which is the molecular nature of the signal detected by DesK and how this protein is able to integrate it in order to further transduce it. On the other hand, FapR is a protein that represses the transcription of almost all the genes encoding the enzymes of fatty acid synthesis. We have solved the 3D structure of one of its domains, proving that it directly binds the positive effector: malonyl-coenzymeA. Our current hypothesis puts forward a major conformational rearrangement triggered by malonyl-CoA, ultimately resulting in FapR releasing its DNA operator. Given that FapR is a global regulator highly conserved among Gram+ bacteria, validation of our structural hypotheses will also contribute to the identification and design of novel antibiotic molecules. 2-Description of the project, methodology : All the target proteins of the project are available from the coordinator's team as recombinant soluble material, highly purified and corresponding both to the full-length and the separated sub-domain versions. We will use an approach combining disciplines coming from biology and physics. We will rely on crystal-state X-ray diffraction to pursue our work aimed at solving the 3D structures of each different component, free or complexed with its cognate partner/ligand: full-length DesK (including its trans-membrane domain), alone and associated with DesR; full-length FapR with and without DNA and/or malonyl-CoA. Detailed comparison of high resolution structures along the transduction pathways will generate and/or validate mechanistic hypotheses guiding to be tested by site-directed mutagenesis and functional experiments. The 'static' crystallographic structures will be complemented by physics techniques that study the proteins in solution. NMR spectroscopy and complete thermodynamic characterizations of the protein targets represent high priority tasks. Methodological developments are envisaged both in NMR analyses of conformational dynamics as well as in structural thermodynamics to explain the cooperative phenomena. We plan to apply structural bioinformatics methods to both systems under study, in order to design small molecules using the structural and dynamic information that will be gathered. We are particularly interested in identifying malonyl-CoA mimetics unable to trigger transcriptional induction. The synthesis of candidate molecules selected from the virtual screenings, will allow their characterization in vitro and their validation in vivo. 3-Expected results : The 3D structure of full-length DesK is a challenging aim that will have a great impact in the understanding of the molecular features of the signal and how DesK integrates it to trigger its His-kinase activity. Studies of DesK-DesR association will shed light on down-stream elements, precisely coordinated at the molecular level with signal sensing. Concerning FapR, our work will allow the experimental validation of the current hypothesis of signal transduction through conformational rearrangement. We will focus on the effects triggered by the signal molecule and the immediate subsequent events that allow FapR to transmit this information. Full-length FapR, alone and in complex with its specific ligands will be extremely valuable to this aim. Finally, the identification of molecules that inhibit the association of FapR and DesK with their specific ligands, will allow us to validate the structural hypotheses as well as to obtain new antibiotics for Gram+ bacteria.