Structural Studies of P-Type ATPases

描述（由申请人提供）：Kdp是一种atp驱动的K+泵，来自细菌和古细菌，是p型atp酶家族的原始成员。像所有p型atp酶一样，Kdp在维持细胞内离子浓度、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方面发挥重要作用。Kdp具有独特的结构，由四个亚基（KdpFABC）组成，其中K+转运和ATP水解的关键机制元件分别被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，这些元素被整合到单个多肽链中。另外的Kdp亚基（KdpC和KdpF）是单通道膜蛋白，类似于真核p型atp酶的调控元件，如磷蛋白、肌磷脂和肌蛋白。本应用程序旨在通过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，它们产生了超过3.5¿分辨率的x射线衍射，以及Kdp的另一种构象的二维晶体，这些晶体已通过冷冻电镜成像。我们还建立了atp酶活性和K+转运的功能测定，这将用于评估位点定向突变的功能影响。在目标1中，我们将研究Kdp复合物的结构和亚基接触的功能相关性。我们将首先着重于通过x射线晶体学获得Kdp配合物的原子结构。我们的主要策略是使用硒代蛋氨酸晶体来代替SAD相位，我们将使用各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。为了评估功能相关性

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.