Molecular Mechanisms of Ion Transport

离子传输的分子机制

• 批准号: 10330684
• 负责人: David L. Stokes
• 金额: $ 25.87万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330684
• 关键词: ATP phosphohydrolase; Address; Adopted; Architecture; Behavior; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biophysics; Cartoons; Cations; Cells; Coupling; Cryoelectron Microscopy; Diffusion; Elements; Family member; Goals; Homeostasis; Hybrids; In Vitro; Individual; Investigation; Ion Transport; Ions; Laboratories; Light; Membrane; Molecular; Molecular Conformation; Molecular Machines; Na(+)-K(+)-Exchanging ATPase; Nature; Pathway interactions; Potassium Channel; Process; Protein Dynamics; Protein Family; Proteins; Protons; Pump; Reaction; Role; Site; Structure; Substrate Specificity; System; Thermodynamics; Transition Elements; Transport Process; Travel; Work; Zinc; animation; antiporter; biochemical tools; biophysical tools; interest; mutant; novel; novel strategies; pressure; simulation; single-molecule FRET

My laboratory is interested in fundamental molecular mechanisms by which cells maintain ionic homeostasis. We study two particular systems responsible for transport of K+ and Zn2+, respectively. Overall goals are the same for both systems, which are to develop a comprehensive understanding of transport from structural as well as thermodynamic perspectives. We will use a broad spectrum of biophysical and biochemical approaches, including cryo-EM for structure determination, in vitro biophysical assays for functional characterization, single-molecule FRET and Molecular Dynamic simulations for analyzing dynamics of the molecules. In this way, we aim to define an energy landscape for each system, annotated with the experimental structures for stable intermediates as well as an appreciation for the high-energy transition states that define the transport pathway. We also seek to understand determinants of substrate specificity and structural elements responsible for the allosteric coupling that underlies energy coupling and regulatory mechanisms. The first system under investigation is KdpFABC, an interesting and unusual hybrid between an ATP-dependent pump related to P-type ATPases and a K+ channel related to the Superfamily of K+ Transporters. Our previous work has defined the architecture of this heterotetramer and suggests a highly novel mechanism for transport, in which K+ enters the selectivity filter of the channel-like subunit, travels 40 Å through a membrane-embedded tunnel, and is then expelled by the pump-like subunit in an energy-dependent manner. We now plan functional analyses of site-directed mutants to validate this hypothesis and to adopt new approaches to study the energetics. The second system is YiiP, a Zn2+/H+ antiporter from the Cation Diffusion Facilitator superfamily. Members of this family form homodimers, have multiple ion binding sites and are thought to function via an alternating access mechanism. For this system, we have characterized two different conformations by cryo-EM analysis and MD simulation, and have identified a role for Zn2+ binding on the transition. We seek to understand better the role of individual Zn2+ binding sites, the nature of occluded states, the transition between inward- and outward-facing states and the role of protons in this process. We plan also to study additional members of this family to address the basis for substrate specificity and structural features that have been postulated to regulate the transport process. In addition to shedding light on these two specific transport mechanisms, we hope that our work will offer new ways to think about transport that goes beyond cartoons and structural animations to incorporate protein dynamics and mapping of the energy landscape to describe the behavior of these molecular machines.

我的实验室对细胞维持离子稳态的基本分子机制很感兴趣。 我们研究了两个特殊的系统负责运输的K+和Zn 2+，分别。总体目标是 这两个系统是一样的，这是发展一个全面的了解交通从结构， 以及热力学的观点。我们将使用广泛的生物物理和生物化学 方法，包括用于结构测定的冷冻EM，用于功能测定的体外生物物理测定， 表征，单分子FRET和分子动力学模拟，用于分析 分子。通过这种方式，我们的目标是为每个系统定义一个能源景观， 稳定中间体的实验结构以及对高能过渡态的评价 定义了运输途径。我们还试图了解底物特异性的决定因素， 负责变构偶联的结构元件，其是能量偶联和调节的基础。 机制等正在研究的第一个系统是KdpFABC，这是一个有趣而不寻常的混合体， 与P型ATP酶相关的ATP依赖性泵和与K+超家族相关的K+通道 传送器。我们以前的工作已经定义了这种异源四聚体的结构，并提出了一个高度的 一种新的运输机制，其中K+进入通道样亚基的选择性过滤器， 通过膜嵌入的隧道，然后由泵样亚单元以能量依赖性排出。 方式我们现在计划对定点突变体进行功能分析，以验证这一假设，并采用新的 研究能量学的方法。第二个系统是YiiP，一个来自阳离子扩散的Zn 2 +/H+反向转运蛋白。 促进者超家族。该家族的成员形成同源二聚体，具有多个离子结合位点， 被认为通过交替访问机制起作用。对于这个系统，我们描述了两个不同的 构象的冷冻-EM分析和MD模拟，并确定了锌2+结合的作用， 过渡我们试图更好地理解单个Zn 2+结合位点的作用，封闭状态的性质， 向内和向外状态之间的过渡以及质子在这一过程中的作用。我们也计划 研究该家族的其他成员，以解决底物特异性和结构特征的基础 被认为是调节运输过程的。除了揭示这两个具体的 我们希望我们的工作将提供新的方式来思考交通， 卡通和结构动画，将蛋白质动力学和能量景观的映射， 描述这些分子机器的行为。