Structure, function and dynamics of the cation-proton antiporter Vc-NhaP2 in Vibrio cholerae

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* **In dangerous human pathogen Vibrio cholerae, as in all marine-borne bacteria, both their survival and dissemination in diverse ecological niches is strictly dependent upon the circulation of alkali cations, primarily Na+ and K+. The membrane of V. cholerae contains a trio of cation-proton antiporters of a specific type, NhaP, that are critical for the homeostasis of K+ and Na+. ******This proposal is focused on the detailed biochemical and physiological analysis of Vc-NhaP2 assisted by its in silico structural modeling. To this end, we propose extensive mutational analysis of Vc-NhaP2 in order to identify the specific atomic groups responsible for the transport of alkali cations. Targets for mutagenesis will be selected based on the model-predicted configuration of the ion-binding site(s) of Vc-NhaP2. Effects of introduced mutations on the activity of Vc-NhaP2 will be measured in sub-bacterial membrane vesicles. Assessment of the acid tolerance response of the NhaP-negative V. cholerae cells carrying mutated Vc-NhaP2 variants expressed from plasmids will be conducted. For the subsequent studies requiring purified protein, tagged versions of Vc-NhaP2 will be constructed, their over-expression and purification will be performed. Purified protein will be subjected to a battery of tests, including (but not limited to) standard biophysical tests for its folding status (CD, Trp fluorescence), reconstitution into proteoliopsomes, NMR assays to gather structural information, and crystallization trials.******In the proposed research, we plan to combine functional analysis based on mutagenesis with the structural in silico modeling. It is expected to result in a significant economy of both time and efforts as well as enhance interpretation of obtained results. To the best of my knowledge, we are the only Canadian research group focusing on antiporters of NhaP type, which, as it becomes more and more evident, play an important role in overall bacterial physiology.******Structural and physiological information gathered in this study will shed a new light on the molecular dynamics and events in the active site of NhaP-type antiporters. It will also be important form the practical point of view, as Vc-NhaP1,2,3 antiporters are likely important for passing the acidic gastric barrier at the early stages of infection caused by many Vibrios, all of them (especially Vc-NhaP2) could provide excellent targets for the development of novel antimicrobial agents to treat strains developing multi-drug resistance (MDR) towards conventional antibiotics. This would be important, for example, for the Canadian fish-farming industry, where anti-Vibrio protection is one of the most important objectives. Structural information gathered in the course of this study