Structural organization and dynamic analyses of Tol membrane proteins – SODATOL

The Tol system located in the bacterial envelope is conserved in Gram negative bacteria. This system is composed of three inner membrane interacting proteins, TolA-TolQ-TolR, one periplasmic TolB protein and an outer membrane anchored Pal lipoprotein. This system maintaining outer membrane integrity has been recently found involved in the late stages of the cell division process. Various protein interactions discovered actually indicated that Tol proteins form complex network linking the inner, outer membranes and the PG. The interaction of TolA C-terminal domain (TolAc) with Pal was demonstrated only in the presence of the proton motive force. The dynamic assembly of the Tol-Pal proteins associated or not with the PG is actually unknown. We have previously proposed a model for the inner membrane TolA-TolQ-TolR helix organization. In this model, residues located in the transmembrane segments (TMS) of TolQ-TolR are suspected to form an ionic channel while small lateral chain residues are involved in TMS assembly. We have demonstrated that the interaction of TolAc with Pal requires the presence of the TolQ-TolR proteins and we have suspected that ion transit through a putative TolQ-TolR channel may induce conformational changes which in turn are transmitted through interactions of Tol TMS to TolA TMS and to the periplasmic region of TolA, thus allowing its interaction with Pal. Such a dynamic long distance energy transduction process is suspected in the energy consuming active transport of Fe3+-siderophores mediated by the TonB system. According that cross-talk between Tol and TonB systems exists and that TMS contain number of conserved residues, our project is an actual challenge devoted to understand the energy transduction mechanisms. The present ANR application associates microbiologists, biochemists and physicians. Our team alone applies for this project. Our group will perform genetic, molecular biology experiments, adjustments of expression and protein purifications and biochemical analyses of complex formation. Moreover, we will use electronic microscopy available in the campus to get image analyses on the purified complexes. It is important to indicate that we recently published new mutations in TolQ-TolR TMS that map residues located in the aqueous ion channel and further define TMS organization. Moreover, our last published results have revealed dynamic movement of the periplasmic region of TolR in relation with pmf and Tol functioning. Results in progress concern the analyses of each residue present in all the Tol TMS. One associated team will crystallize Tol membrane proteins (obtained from various Gram negative bacteria) and complexes to get structural resolutions. One important point concerns the purification of proteins and structural analyses that will be performed by Nadir Seddiki in the laboratory of Dr Susan Buchanan. Nadir has just started his PhD in the University of the Mediterranean with a NIH-PhD fellowship in co-direction with Susan Buchanan (NIH, NIDDK, Bethesda) and Roland Lloubes. It is noticeable to specify that Dr Susan Buchanan team is working on the TonB system and on the structure of membrane proteins. Then, in this perspective our results on membrane Tol multimeric complexes obtained by tandem cystéine scanning will be useful to stabilize Tol protein complexes. In association with physician teams, we will check Electronic Paramagnetic Resonance (EPR) to analyze dynamic motions of membrane Tol protein complexes. According to our actual systematic cysteine scanning experiments, we will posses new information on accessible and non-interfering residue required to further label with EPR probes to analyze dynamic motions of Tol inner membrane complexes. The EPR associated team (not participant) is directed by Bruno Guigiarelli and we will collaborate with Valerie Belle and André Fournel. Eric Cascales in our team will also advise this project (not listed since he applies for the ANR 'jeunes chercheurs'). In conclusion, this projet which associates microbiologists, biochemists and physicians is focussed on the inner membrane Tol protein complexes and will issue in the understanding on the dynamic and structural data on protein complexes and key residues required for complex functioning and assembly. In addition, our results will give new lighting on long distance energy transducing nanomachines.