Elucidation of the Assembly of a Tripartite Multidrug Pump. (360G-Wellcome-084821_Z_08_Z)

Our programme will build upon our studies of the MtrCDE multi-drug pump from Neisseria gonorrhoeae. We have overproduced MtrC, D and E; and established via pull-down assays, chemical cross-linking and microcalorimetry that MtrD and MtrE can interact with MtrC and with one another. Our aims are to map the sites of interaction by proteolytic-fragmentation and mass-spectrometry fingerprinting of the cross-linked proteins. This will enable us to model the docking of the proteins as a guide to the in sertion of cysteines for cross-linking to stabilise the ternary-complex for imaging by AFM. Ultimately, our aim is to build an atomic-resolution model of the assembly by docking the high-resolution structures of the individual proteins into the AFM topographs. Towards this end, we have already obtained crystals of each protein that diffract to around 7-8 that will be further optimized for high-resolution diffraction. As an alternative strategy we will undertake the crystallisation and structure determination of the binary- and ternary-complexes. We will utilise our docking models and/or structures to identifying residues that can stabilize the ternary-complex; mutate these residues and test for pump assembly (via pull-down assays, microcalorimetry and by monitoring the interaction of fluorophore-labelled proteins by fluorescence-energy-transfer) and function (via resistance assays).