Structural Studies of P-Type ATPases

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、建立用于二次运输过程的膜梯度以及在细菌中的膨胀和细胞形状方面发挥着重要作用。 Kdp 具有由四个亚基 (KdpFABC) 组成的独特结构，其中 K 运输和 ATP 水解的关键机制元件被分离到不同的亚基上：分别是 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，例如受磷蛋白、肌磷脂和肌色素。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，它们产生了超过 3.5 ¡ 分辨率的 X 射线衍射，以及采用冷冻电镜成像的替代构象的 Kdp 二维晶体。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影响。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。我们首先将重点关注通过 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取代的晶体进行 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有序度。为了评估功能相关性 在此结构中看到的亚基相互作用，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，并解决运输过程中各个亚基相互作用是动态还是静态的问题。我们将使用 ATP 酶活性和 K 转运分析来研究目标 1 中确定的突变体中的能量耦合。我们还将使用冷冻电镜从 2D 膜结合晶体中确定 Kdp 的结构。结晶条件表明，X 射线得出的 3D 晶体结构和冷冻电镜得出的 2D 晶体结构将代表反应循环的替代构象。这项工作将测试两个主要假设：KdpB 中依赖于 ATP 的构象变化与 KdpA 中的离子门物理耦合，以控制 K 传输；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。

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.