Structural and functional studies of homologous membrane molecular motors – MeMoX

The aim of this project is to decipher the structure and function of the Tol and Ton complexes present in various Gram-negative bacteria, two membrane proteins complexes that transmit energy across the cell envelope. These complexes are involved in essential functions such as nutrient importation, cell division and the integrity of the outer membrane. The Tol and Ton inner membrane complexes harbour respectively the TolQ/TolR/TolA and and ExbB/ExbD/TonB subunits. The TolQ/TolR and ExbB/ExbD motors harvest the energy derived from the proton motive force to create mechanical movement that are transmitted to their associated proteins TolA and TonB, respectively. TolA and TonB are elongated proteins that span the periplasmic space and interact with OM proteins, the TolB-Pal complex and the TonB dependent receptors, respectively. Recently, thanks to an ANR funding (BacMolMot) we have solved the high-resolution structure of the membrane ExbB/ExbD motor complex. Surprisingly, the complex is formed by a bulky cytoplasmic alpha-helical ExbB pentamer containing a centralized transmembrane pore that is occluded by one ExbD molecule. However, the structure of the ExbD oligomer present in the complex is not totally solved. The main purpose of this project is to determine if the Tol and Ton molecular motors from various bacteria share a similar structure and/or operate differently. In addition, we will analyse if functional cross-talk could occur in E. coli complemented with heterologous TolQ/TolR and ExbB/ExbD complexes. To address how these motors operate we will study the role of the TolQ/TolR motor on the mode of action of the Tol-Pal complex during E. coli cell division.
Our project will focus on four mains topics covering a multi-disciplinary approach involving genetic, biochemical, biophysical and epifluorescence microscopy approaches: 1) Based on our expertise in membrane protein purification, we plan to overproduced and purified ExbB/ExbD and TolQ/TolR molecular motors from various bacterial genus. This task will be performed by the team of Roland Lloubes. 2) Determination of the 3D structure of the different complexes will be performed in a continuing collaboration with the team of Susan Buchanan (NIH, Bethesda). 3) Parallel to this structural approach, we will study the role of Tol-Pal system in the late stages of cell division by in situ fluorescence microscopy in collaboration with the team of Tâm Mignot (LCB, Marseille). We have already engineered strains producing GFP or mCherry fused to the Tol-Pal proteins which remain totally functional. All these chromosomal constructs were introduced at the native, original loci. We plan to test the cellular location of each hybrid fluorescent protein in different tol-pal deletion/mutation background to get valuable information about the sequential recruitment of the Tol-Pal proteins at the septum, their respective importance and will elucidate the role of the TolQ/TolR motor in the Tol-Pal assembly. We also plan to develop a microfluidic approach to study the dynamic of the Tol-Pal proteins in response to various growth conditions by high resolution time lapse microscopy. 4) Finally, interchangeability of the two molecular motors TolQ/TolR and ExbB/ExbD will be check for both Tol and Ton functions using phenotypic and microscopy analysis. This task will be performed in collaboration with the team of TM.
The RL team, involved in each task, will coordinate a new national collaboration initiated recently with the team of TM to elucidate the role of the TolQ/TolR motor in the Tol-Pal assembly and will strengthen the international collaboration managed previously with SB team (with whom we solved the 3D structure of the E. coli ExbB/ExbD motor).