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（由适用提供）：KDP是来自ATP驱动的K+泵，来自细菌和考古学，是P型ATPases家族的原始成员。像所有P型ATPases一样，KDP在维持细胞内离子浓度和建立用于二级运输过程的膜梯度以及用于Turgor和细胞形状的细菌中具有重要作用。 KDP具有独特的架构，由四个亚基（KDPFABC）组成，其中K+传输和ATP水解的关键机械元素分别隔离到不同的亚基上：KDPA和KDPB。该体系结构与其他P型ATPases显着对比，其中这些元素被整合到单个多肽链中。附加的KDP亚基（KDPC和KDPF）是单次膜蛋白，类似于真核P型ATPases的调节元件，例如磷脂，Sarcolipin和Sarcolemman。该应用程序旨在定义KDP的能量耦合的结构和机械基础。初步结果包括KDP复合物的两种晶体形式，它们在替代构象中产生了X射线衍射超过3.5»kDP的分辨率和2D晶体，这些构型已被冷冻EM成像。我们还建立了用于ATPase活性和K+转运的功能测定，该测定法将用于评估位置定向突变的功能效应。对于AIM 1，我们将研究KDP复合体的架构以及亚基触点的功能相关性。我们最初将专注于通过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蛋氨酸取代的晶体进行SAD阶段，我们将使用各种方法来改善现有晶体形式的衍射分辨率，包括优化的Purificaton和添加配体以增加晶体顺序的条件。为了评估 在该结构中看到的亚基相互作用，我们将进行突变以在亚基界面上生存，并使用基于细胞的测定法测试所得KDP突变体的生存能力。我们还将将半胱氨酸保留在应用界面的侧面上，并测试能够交联亚基的能力。对于AIM 2，我们将研究KDP中的构象变化，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。我们将使用测定ATPase活性和K+传输来研究AIM 1中确定的突变体中的能量耦合。我们还将使用Cryo-EM来确定来自2D，膜结合的晶体的KDP结构。结晶条件表明，通过X射线和Cryo-EM的2D晶体来自3D晶体的结构将代表相对于反应周期的替代会议。这项工作将检验两个主要假设：KDPB中的ATP依赖性会议变化在物理上与KDPA中的离子门耦合以控制K+传输，并且KDPC和KDPF亚基与KDPB相互作用并控制其构象变化。鉴于KDPA与次级转运蛋白和K+通道的相似性，这项工作还将有助于定义泵，转运蛋白和通道之间的机械边界和进化关系。