Structural Studies of P-Type ATPases

描述(申请人提供)：KDP是一种由细菌和古生菌中的ATP驱动的K泵，是P型ATPase家族的原始成员。像所有的P型ATPase一样，KDP在维持细胞内离子浓度和建立用于二次运输过程的膜梯度以及在细菌中用于肿胀和细胞形状方面具有重要作用。KDP有一个独特的结构，由四个亚基(KdpFABC)组成，其中K运输和ATP水解的关键机制元件分别被分离到不同的亚基：KdpA和KdpB。这种结构与其他P型ATPase明显不同，在P型ATPase中，这些元件被整合到一个单一的多肽链中。附加的KDP亚基(KdpC和KdpF)是一种单通道膜蛋白，类似于真核细胞P型ATPase的调节元件，如磷蛋白、肌磷脂和肌联蛋白。本申请旨在定义KDP能量耦合的结构和机械基础。初步结果包括两种晶体形式的KDP络合物，它们产生了分辨率超过3.5°的X射线衍射，以及由Cryo-EM成像的交替构象的KDP的2D晶体。我们还建立了ATPase活性和K转运的功能分析，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复合体的架构和亚单位接触的功能相关性。我们首先将重点放在通过X射线结晶学获得KDP络合物的原子结构上。我们的主要策略是使用硒蛋氨酸取代的晶体来进行SAD相变，我们将使用各种方法来提高现有晶型的衍射分辨率，包括优化提纯条件和添加配体以增加晶体有序性。为了评估功能相关性， 在这个结构中看到的亚基相互作用，我们将对亚单位界面的残基进行突变，并使用基于细胞的分析来测试得到的KDP突变体的生存能力。我们还将把半胱氨酸残基放在界面的相对两侧，并测试亚基的交联性。对于目标2，我们将研究KDP的构象变化，并解决个别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ATPase活性和K转运的分析来研究Aim 1中确定的突变体中的能量耦合。我们还将使用冷冻-EM来从2D膜结合晶体中确定KDP的结构。结晶条件表明，由X射线得到的3D晶体和由Cryo-EM得到的2D晶体的结构将在反应周期中代表不同的构象。这项工作将检验两个主要假设：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的运输，以及KdpC和KdpF亚基与KdpB相互作用并控制其构象变化。鉴于KdpA与次级转运体和K通道的相似性，这项工作还将有助于确定泵、转运体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.