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 是一种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方面具有重要作用。 ，并建立用于二次运输过程的膜梯度，以及在细菌中用于膨胀和细胞形状的 Kdp 具有由四个亚基组成的独特结构。 (KdpFABC)，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上：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。额外的 Kdp 亚基（KdpC 和 KdpF）是单程膜蛋白，类似于真核 P 型 ATP 酶的调节元件，例如该应用旨在定义 Kdp 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，其产生的 X 射线衍射超过 3.5 ¿我们还建立了 ATP 酶活性和 K+ 转运的功能测定，用于评估定点突变对 Aim 1 的功能影响。 ，我们将研究 Kdp 复合物的结构和亚基接触的功能相关性。我们首先将重点关注通过 X 射线晶体学获得 Kdp 复合物的原子结构。硒代甲硫氨酸取代晶体用于 SAD 定相，我们将使用多种方法来提高现有晶体形式的衍射分辨率，包括优化纯化条件和添加配体以增加晶体顺序，以评估其功能相关性。 为了在该结构中看到亚基相互作用，我们将对亚基界面上的残基进行突变，并使用基于细胞的测定法来测试所得 Kdp 突变体的活力。我们还将在界面的相对侧放置半胱氨酸残基并进行测试。对于目标 2，我们将研究 Kdp 的构象变化并确定各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+ 运输的测定来研究能量。目标 1 中确定的突变体中的耦合。我们还将使用冷冻电镜从 2D 膜结合晶体确定 Kdp 的结构。结晶条件表明，通过 X 射线确定 3D 晶体的结构，通过冷冻电镜确定 2D 晶体的结构。将代表与反应循环相关的替代构象。这项工作将测试两个主要假设：KdpB 中的 ATP 依赖性构象变化与 KdpA 中的离子门物理耦合以控制。鉴于 KdpA 与二级转运蛋白和 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。