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

** *在危险的人类病原体霍乱弧菌中，如同在所有海洋传播的细菌中一样，它们在不同生态位中的生存和传播都严格依赖于碱阳离子的循环，主要是Na+和K+。霍乱弧菌的膜含有三种特定类型的阳离子-质子反转运蛋白NhaP，它们对K+和Na+的稳态至关重要。******本提案的重点是Vc-NhaP2的详细生化和生理分析，协助其在硅结构建模。为此，我们建议对Vc-NhaP2进行广泛的突变分析，以确定负责碱阳离子运输的特定原子群。突变目标将根据模型预测的Vc-NhaP2离子结合位点的配置来选择。引入突变对Vc-NhaP2活性的影响将在亚细菌膜囊泡中测量。将对携带质粒表达的Vc-NhaP2突变体的nhap阴性霍乱弧菌细胞的耐酸反应进行评估。对于后续需要纯化蛋白的研究，将构建标记版本的Vc-NhaP2，并进行过表达和纯化。纯化后的蛋白质将进行一系列测试，包括（但不限于）标准的生物物理测试，以检测其折叠状态（CD，色氨酸荧光），重组成蛋白质体，NMR分析以收集结构信息，以及结晶试验。******在提出的研究中，我们计划将基于诱变的功能分析与结构硅建模相结合。预计这将大大节省时间和精力，并加强对已获得结果的解释。据我所知，我们是加拿大唯一一个专注于NhaP型反转运蛋白的研究小组，随着NhaP型反转运蛋白越来越明显，在整个细菌生理中发挥着重要作用。******本研究收集的结构和生理信息将为nhap型反转运蛋白活性位点的分子动力学和事件提供新的线索。从实际应用的角度来看，Vc-NhaP1,2,3对转运蛋白在许多弧菌感染的早期阶段可能对通过酸性胃屏障很重要，所有这些（特别是Vc-NhaP2）都可以为开发新型抗菌药物提供良好的靶点，以治疗对常规抗生素产生多重耐药（MDR）的菌株。例如，这对加拿大养鱼业来说是很重要的，在那里，抗弧菌保护是最重要的目标之一。本研究过程中收集的结构信息

* **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