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，就像在所有海洋传播的细菌中一样，它们在不同的生态位中的生存和传播都严格依赖于碱性阳离子的循环，主要是钠和钾。霍乱弧菌的膜含有一种特殊类型的阳离子-质子逆向转运蛋白NhaP，它对K和Na的动态平衡至关重要。 本研究主要针对VC-NhaP2进行了详细的生化和生理分析，并辅以其电子结构模拟。为此，我们建议对VC-NhaP2进行广泛的突变分析，以确定负责碱性阳离子运输的特定原子基团。突变的目标将根据模型预测的VC-NhaP2的离子结合位点(S)的配置来选择。引入的突变对VC-NhaP2活性的影响将在亚细菌膜泡中进行测量。将评估携带从质粒表达的突变VC-NhaP2变异体的NhaP阴性霍乱弧菌细胞的耐酸反应。对于需要纯化蛋白的后续研究，将构建VC-NhaP2的标记版本，并进行其过表达和纯化。纯化的蛋白质将接受一系列测试，包括(但不限于)其折叠状态的标准生物物理测试(CD、Trp荧光)、重组为蛋白脂体、收集结构信息的核磁共振分析以及结晶试验。 在拟议的研究中，我们计划将基于突变的功能分析与电子计算机建模中的结构相结合。预计这将大大节省时间和精力，并加强对所取得成果的解释。据我所知，我们是加拿大唯一一个专注于NhaP类型的抗转运蛋白的研究小组，随着它变得越来越明显，它在整个细菌生理学中扮演着重要的角色。 这项研究收集的结构和生理信息将为了解NhaP型逆向转运蛋白活性部位的分子动力学和事件提供新的线索。从实用的角度来看，这也将是重要的，因为VC-NhaP1，2，3逆向转运体在许多弧菌感染的早期阶段可能是通过酸性胃屏障的重要因素，所有这些(特别是VC-NhaP2)都可以为治疗对常规抗生素产生多药耐药(MDR)的菌株提供极好的靶点。例如，这对加拿大养鱼业将是重要的，因为在该行业中，抗弧菌保护是最重要的目标之一。在本研究过程中收集的结构性信息

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