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，在所有的海洋传播的细菌，它们的生存和传播在不同的生态位是严格依赖于循环的碱金属阳离子，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向转运蛋白NhaP，其对K+和Na+的稳态至关重要。 该提案的重点是详细的生化和生理分析的Vc-NhaP 2辅助其在计算机结构建模。 为此，我们提出了广泛的突变分析的Vc-NhaP 2，以确定负责运输的碱金属阳离子的特定原子团。诱变的靶标将基于模型预测的Vc-NhaP 2的离子结合位点的构型来选择。 将在亚细菌膜囊泡中测量引入的突变对Vc-NhaP 2活性的影响。 将对携带由质粒表达的突变Vc-NhaP 2变体的NhaP阴性霍乱弧菌细胞的酸耐受性反应进行评估。 对于需要纯化蛋白的后续研究，将构建标记形式的Vc-NhaP 2，将进行其过表达和纯化。 纯化的蛋白质将接受一系列检测，包括（但不限于）其折叠状态（CD、Trp荧光）的标准生物物理学检测、蛋白脂质体重构、收集结构信息的NMR测定和结晶试验。 在拟议的研究中，我们计划将基于诱变的联合收割机功能分析与结构计算机建模相结合。 预计这将大大节省时间和精力，并加强对所获结果的解释。据我所知，我们是加拿大唯一一个专注于NhaP类型反向转运蛋白的研究小组，随着它变得越来越明显，在整个细菌生理学中发挥着重要作用。 在这项研究中收集的结构和生理信息将揭示一个新的光的分子动力学和事件的活性位点的NhaP型反向转运蛋白。从实践的角度来看，这也是重要的，因为Vc-NhaP 1，2，3反向转运蛋白对于在由许多弧菌引起的感染的早期阶段通过酸性胃屏障可能是重要的，所有这些（特别是Vc-NhaP 2）都可以为开发新的抗微生物剂以治疗对常规抗生素产生多药耐药性（MDR）的菌株提供极好的靶标。例如，这对加拿大的养鱼业很重要，因为抗弧菌保护是该行业最重要的目标之一。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