Investigation of Alkali/H+ Antiport by NapA Using Multiscale Simulation Methods

 描述（由申请人提供）：Na+/H+反向转运蛋白是生物学上普遍存在的一类蛋白质，通过催化内部Na+（或Li+）交换外部H+来维持细胞稳态。在人类中，它们的功能障碍与癌症以及心脏、血管、胃和肾脏疾病有关;在植物中，它们的表达与耐盐性有关;在细菌中，它们的活性与毒力有关。由于大量的Na+/H+反向转运蛋白（包括人类中的至少11种亚型），设计针对这些蛋白质的药物的努力面临着选择性地靶向许多密切相关的同源物中的一种的困难挑战。这项任务需要对反向转运蛋白的结构、功能和能量学有非常详细的了解;然而，Na+/H+反向转运蛋白仍然不完全了解。碱和H+运输的机制是有争议的，这些过程背后的自由能分布尚未阐明，这些过程对碱阳离子身份和外部pH值的依赖需要解释。 最近，对细菌Na+/H+逆向转运蛋白NapA的晶体结构进行了研究。嗜热菌，以3 μ m分辨率解析。与之前对其他Na+/H+反向转运蛋白的研究相反，该晶体 结构的开放（主动）的形式解决，提供了一个无与伦比的质量的起点，详细的理论研究的机制和热力学的antiport。 NapA功能已被提出依赖于碱和H+结合到关键活性位点残基之间的耦合，以及大于10 μ m的大规模域运动，其交替地将结合位点暴露于细胞内部和外部。因此，描述反向端口过程将需要使用桥接各种时间和长度尺度的方法，包括粗粒度模型（例如，弹性网络模型），增强的采样方法（例如，马尔可夫状态模型和元自洽模型）、经典分子动力学（MD）、反应MD和量子力学/分子力学方法。为了与实验建立联系，这些方法将被应用于研究野生型NapA和一些突变体的反向转运的能量学的阳离子和pH依赖性。这项工作将提供反向转运过程的详细描述，揭示NapA重要结构元件的作用及其对各种盐和pH条件的响应，从而深入了解大量同源蛋白的行为。

 DESCRIPTION (provided by applicant): Na+/H+ antiporters are a biologically ubiquitous class of proteins that maintain cell homeostasis by catalyzing the exchange of internal Na+ (or Li+) for external H+. In humans, their dysfunction has been associated with cancer and with cardiac, vascular, gastric, and kidney diseases; in plants, their expression has been correlated with salt resistance; and in bacteria, their activity has been related to virulence. Due to the large number of Na+/H+ antiporters (including at least eleven sub-types in humans), efforts to design drugs for these proteins face the difficult challenge of selectively targeting just one of a number of closel related homologues. This task requires a highly detailed understanding of antiporter structure, function, and energetics; however, Na+/H+ antiporters remain incompletely understood. The mechanisms of alkali and H+ transport are debated; the free energy profiles underlying these processes have not been elucidated; and the dependence of these processes on alkali cation identity and external pH require explanation. Recently, the crystal structure of a bacterial Na+/H+ antiporter, NapA from T. thermophilus, was solved at 3 Å resolution. In contrast to previous efforts on other Na+/H+ antiporters, the crystal structure was solved in the open (active) form, providing a starting point of unparalleled quality for a detailed theoretical investigation of the mechanism and thermodynamics of antiport. NapA function has been proposed to depend on the coupling between alkali and H+ binding to key active site residues, and large-scale domain motions of more than 10 Å that alternately expose the binding site to the cell interior and exterior. Therefore, describing the antiport process will require the use of methods that bridge a variety of time and length scales, including coarse-grained models (e.g., elastic network models), enhanced sampling methods (e.g., Markov state models and metadynamics), classical molecular dynamics (MD), reactive MD, and quantum mechanics/molecular mechanics methods. In order to forge connections with experiment, these methods will be applied to study the cation- and pH-dependence of the energetics of antiport for both wild-type NapA and a number of mutants. This work will provide a detailed picture of the antiport process, revealing the role of NapA's important structural elements and its response to various salt and pH conditions, and thereby providing insights into the behavior of a large number of homologous proteins.