Structural Studies of P-Type ATPases

P 型 ATP 酶的结构研究

• 批准号: 8712800
• 负责人: David L. Stokes
• 金额: $ 32.21万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8712800
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Architecture; Bacteria; Beryllium; Binding; Biological Assay; Ca(2+)-Transporting ATPase; Cell Shape; Cell membrane; Cells; Complex; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Cysteine; Elements; Escherichia coli; Eukaryotic Cell; Family; Family Study; Family member; Goals; Homeostasis; Homologous Gene; Hydrolysis; Image; Individual; Ion Transport; Ions; L-Selenomethionine; Life; Ligands; Light; Membrane; Membrane Proteins; Methionine; Molecular; Molecular Conformation; Mutagenesis; Mutation; Na(+)-K(+)-Exchanging ATPase; Nature; Organism; Phase; Physiological Processes; Potassium; Potassium Channel; Preparation; Proteins; Pump; Reaction; Regulatory Element; Resolution; Roentgen Rays; Role; Secondary to; Side; Site; Solutions; Structure; Testing; Transport Process; Vanadates; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; analog; base; crosslink; improved; insight; mutant; phospholamban; polypeptide; potassium ion; progenitor; public health relevance; sarcolipin; screening; three dimensional structure

DESCRIPTION (provided by applicant): Kdp is an ATP-driven K+ pump from bacteria and archae that is a primordial member of the family of P-type ATPases. Like all P-type ATPases, Kdp has an important role in maintaining intracellular ion concentrations, and establishing a membrane gradient that is used for secondary transport processes, and in bacteria for turgor and for cell shape. Kdp has a unique architecture consisting of four subunits (KdpFABC), in which key mechanistic elements for K+ transport and ATP hydrolysis are segregated onto distinct subunits: KdpA and KdpB, respectively. This architecture contrasts markedly from other P-type ATPases, in which these elements are integrated into a single polypeptide chain. The additional Kdp subunits (KdpC and KdpF) are single-pass membrane proteins that resemble regulatory elements of eukaryotic P-type ATPases, such as phospholamban, sarcolipin and sarcolemman. This application seeks to define the structural and mechanistic bases for energy coupling by Kdp. Preliminary results include two crystal forms of the Kdp complex, which have produced X-ray diffraction beyond 3.5 ¿ resolution and 2D crystals of Kdp in an alternate conformation which have been imaged by cryo-EM. We have also established functional assays for ATPase activity and K+ transport, which will be used to evaluate the functional effects of site-directed mutations. For Aim 1, we will study the architecture of the Kdp complex and the functional relevance of subunit contacts. We will initially focus on obtaining an atomic structure of the Kdp complex by X-ray crystallography. Our primary strategy is to use seleno-methionine substituted crystals for SAD phasing and we will use a variety of approaches to improve the resolution of diffraction from existing crystal forms, including optimized conditions for purificaton and addition of ligands to increase crystal order. In order to evaluate the functional relevance of subunit interactions seen in this structure, we will make mutations to residues at subunit interfaces and use a cell-based assay to test the viability of the resulting Kdp mutants. We will also place cysteine residues on apposing sides of the interface and test for the ability to crosslink the subunits. For Aim 2, we will study conformational changes in Kdp and address whether individual subunit interactions are dynamic or static during transport. We will use assays for ATPase activity and K+ transport to study energy coupling in the mutants identified in Aim 1. We will also use cryo-EM to determine a structure of Kdp from 2D, membrane-bound crystals. Crystallization conditions indicate that structures from 3D crystals by X-ray and from 2D crystals by cryo-EM will represent alternative conformations with respect to the reaction cycle. This work will test two main hypotheses: that ATP- dependent conformational changes in KdpB are physically coupled to ion gates in KdpA in order to control K+ transport, and that KdpC and KdpF subunits interact with KdpB and control its conformational changes. Given the similarity of KdpA with secondary transporters and K+ channels, this work will also help define mechanistic boundaries and evolutionary relationships between pumps, transporters and channels.

描述（由申请人提供）：Kdp是来自细菌和古细菌的ATP驱动的K+泵，是P型ATP酶家族的原始成员。与所有P型ATP酶一样，Kdp在维持细胞内离子浓度、建立用于次级转运过程的膜梯度以及在细菌中用于膨压和细胞形状方面具有重要作用。Kdp具有由四个亚基（KdpFABC）组成的独特结构，其中K+转运和ATP水解的关键机制元件分别分离到不同的亚基：KdpA和KdpB。这种结构与其他P型ATP酶形成鲜明对比，在其他P型ATP酶中，这些元件被整合到单个多肽链中。另外的Kdp亚基（KdpC和KdpF）是类似于真核生物P型ATP酶的调节元件的单程膜蛋白，例如受磷蛋白、肌磷脂和肌膜蛋白。本申请旨在通过Kdp定义能量耦合的结构和机制基础。初步结果包括Kdp复合物的两种晶体形式，其产生的X射线衍射超过3.5 º分辨率，以及Kdp的2D晶体，其处于交替构象，已通过cryo-EM成像。我们还建立了ATP酶活性和K+转运的功能测定，这将用于评估定点突变的功能效应。对于目标1，我们将研究Kdp复合物的结构和亚基接触的功能相关性。我们将首先专注于通过X射线晶体学获得Kdp复合物的原子结构。我们的主要策略是使用硒代甲硫氨酸取代晶体进行SAD定相，我们将使用各种方法来提高现有晶形的衍射分辨率，包括优化纯化条件和添加配体以增加晶体有序度。为了评价功能相关性， 由于在该结构中观察到的亚基相互作用，我们将对亚基界面处的残基进行突变，并使用基于细胞的测定来测试所得Kdp突变体的活力。我们还将半胱氨酸残基放置在界面的相对侧，并测试交联亚基的能力。对于目标2，我们将研究Kdp的构象变化，并解决单个亚基相互作用在运输过程中是动态的还是静态的。我们将使用ATP酶活性和K+转运的测定来研究目的1中鉴定的突变体中的能量偶联。我们还将使用cryo-EM从2D膜结合晶体中确定Kdp的结构。结晶条件表明，从3D晶体通过X射线和从2D晶体通过冷冻EM的结构将代表相对于反应循环的替代构象。这项工作将测试两个主要假设：KdpB中ATP依赖的构象变化与KdpA中的离子门物理耦合以控制K+转运，以及KdpC和KdpF亚基与KdpB相互作用并控制其构象变化。鉴于KdpA与二级转运蛋白和K+通道的相似性，这项工作也将有助于定义泵，转运蛋白和通道之间的机制边界和进化关系。