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感兴趣。 我们研究了分别负责 K+ 和 Zn2+ 运输的两个特定系统。总体目标是 两个系统都是一样的，都是为了从结构到对运输有一个全面的理解 以及热力学观点。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电镜、用于功能性的体外生物物理测定 用于分析动力学的表征、单分子 FRET 和分子动力学模拟 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，并用 稳定中间体的实验结构以及对高能过渡态的认识 定义传输路径。我们还寻求了解底物特异性的决定因素和 负责能量耦合和调节基础上的变构耦合的结构元件 机制。第一个正在研究的系统是 KdpFABC，它是一个有趣且不寻常的混合体 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 运输商。我们之前的工作已经定义了这种异四聚体的结构，并提出了一种高度 新颖的传输机制，其中 K+ 进入通道状亚基的选择性过滤器，行进 40 Å 通过膜嵌入的隧道，然后由类似泵的亚基以能量依赖的方式排出 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，一种来自阳离子扩散的 Zn2+/H+ 反向转运蛋白 促进者超家族。该家族的成员形成同型二聚体，具有多个离子结合位点，并且 认为通过交替访问机制发挥作用。对于这个系统，我们描述了两种不同的特征 通过冷冻电镜分析和 MD 模拟对构象进行了分析，并确定了 Zn2+ 结合在 过渡。我们寻求更好地了解各个 Zn2+ 结合位点的作用、封闭态的性质、 向内和向外状态之间的转变以及质子在这个过程中的作用。我们还计划 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 被假定为规范运输过程。除了阐明这两个具体 运输机制，我们希望我们的工作能够提供新的方式来思考超越运输 卡通和结构动画将蛋白质动力学和能源景观映射结合起来 描述这些分子机器的行为。