Potassium transport by the KdpFABC complex

KdpFABC 复合体的钾转运

• 批准号: 9982340
• 负责人: David L. Stokes
• 金额: $ 34.14万
• 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982340
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Adopted; Affect; Affinity; Animals; Architecture; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Cations; Cell membrane; Cells; Chemicals; Communication; Complex; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Cytoplasm; Elements; Environment; Evolution; Extracellular Fluid; Family; Food; Growth; Homeostasis; Ingestion; Ions; Kinetics; Light; Mass Spectrum Analysis; Measures; Membrane; Membrane Potentials; Molecular Conformation; Mutagenesis; Mutation; Na(+)-K(+)-Exchanging ATPase; Nature; Operon; Organism; Osmoregulation; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphoserine; Phylogenetic Analysis; Physiological; Play; Potassium; Potassium Channel; Process; Property; Protons; Pump; Reaction; Rest; Role; Serine; Structure; System; Testing; Transmembrane Transport; X-Ray Crystallography; base; enzyme activity; extracellular; falls; inhibitor/antagonist; member; mutant; novel; pH Homeostasis; particle; periplasm; plant fungi

Potassium was adopted by the earliest organisms as the most prevalent cation in the cytoplasm. Today, the K+ gradient across the plasma membrane is largely responsible for the resting potential of all cells and high cytoplasmic K+ concentrations are essential for enzyme activity, osmoregulation and pH homeostasis. Animals rely on Na+/K+-ATPase, which is a P-type ATPase to maintains an ~10-fold gradient in K+. Whereas animals ingest K+ rich food and maintain homeostasis of extracellular fluids, plants, fungi and bacteria have to survive in a wide range of environmental conditions which can include limitations in K+. These organisms have evolved different K+ transport systems that are capable of generating gradients between 103 and 105. Transporters with moderate K+ affinity are constitutively expressed and, under normal circumstances, are capable of maintaining these gradients. In order to survive at very low K+ concentrations, however, bacteria have evolved a high- affinity, inducible system that functions as a primary active transporter. In particular, the kdp operon is expressed at micromolar K+ concentrations, producing a heterotetrameric membrane complex called KdpFABC that uses ATP to pump K+ into the cell. This transport system represents an unprecedented partnership between a channel-like subunit (KdpA) and a pump-like subunit (KdpB). The former belongs to the Superfamily of K+ transporters and the latter belongs to the P-type ATPase family. As part of the Kdp complex, both subunits have been repurposed relative to other members of their respective families. In particular, KdpB is a P-type ATPase that does not pump, but rather that uses ATP-driven conformational changes to control KdpA. KdpA has an architecture derived from K+ channels that has been adapted to move ions against an electro- chemical potential. We recently solved the first crystal structure of the KdpFABC complex, which sets the stage for characterizing the elements responsible for this process and for understanding communication and energy coupling between the subunits. Based on this structure, we have developed specific hypotheses which will be addressed through three specific aims. In Aim 1, we will use biochemical and biophysical assays to characterize steps in the reaction cycle and to identify conditions for stabilizing specific reaction intermediates. These assays will be used in conjunction with mutagenesis to identify the gates controlling transport through KdpA and to address mechanisms by which they are coupled to ATP-driven changes in KdpB. In Aim 2, we will use single-particle cryo-EM to solve structures of stabilized reaction intermediates in order to visualize the structural elements that drive transport. In Aim 3, we will address our unexpected finding of an inhibitory phosphoserine on KdpB. The first priority will be to minimize the level of phosphorylation either by mutagenesis, phosphatase treatment or growth conditions; an active complex with minimal phosphorylation is necessary to pursue the first two aims. In addition, we will explore our hypothesis for a physiological role of serine phosphorylation to shut off Kdp activity once extracellular K+ concentrations are restored.

钾被最早的生物体视为细胞质中最普遍的阳离子。今天，K+ 跨质膜的梯度在很大程度上决定了所有细胞的静息电位和高电位 细胞质 K+ 浓度对于酶活性、渗透压调节和 pH 稳态至关重要。动物 依赖 Na+/K+-ATP 酶（一种 P 型 ATP 酶）来维持 K+ 约 10 倍的梯度。而动物 摄入富含 K+ 的食物并维持细胞外液的稳态，植物、真菌和细菌必须生存 适用于各种环境条件，其中可能包括 K+ 的限制。这些生物已经进化了 不同的 K+ 传输系统能够产生 103 到 105 之间的梯度。 中等 K+ 亲和力是组成型表达的，并且在正常情况下能够维持 这些梯度。然而，为了在非常低的 K+ 浓度下生存，细菌进化出了一种高 K+ 浓度的细菌。 亲和力诱导系统，充当主要主动转运蛋白。特别地，kdp操纵子是 以微摩尔 K+ 浓度表达，产生称为 KdpFABC 的异四聚体膜复合物 使用 ATP 将 K+ 泵入细胞。该运输系统代表了前所未有的合作伙伴关系 位于通道状亚基 (KdpA​​) 和泵状亚基 (KdpB) 之间。前者属于超家族 K+ 转运蛋白，后者属于 P 型 ATP 酶家族。作为 Kdp 综合体的一部分，两者 子单元相对于各自家族的其他成员已被重新调整用途。特别地，KdpB 是 P 型 ATP 酶不泵送，而是使用 ATP 驱动的构象变化来控制 KdpA。 KdpA 具有源自 K+ 通道的架构，该架构适用于移动离子对抗电 化学势。我们最近解决了 KdpFABC 复合物的第一个晶体结构，这奠定了基础 用于描述负责此过程的元素以及理解通信和能量 子单元之间的耦合。基于这个结构，我们提出了具体的假设 通过三个具体目标来解决。在目标 1 中，我们将使用生化和生物物理检测来 表征反应循环中的步骤并确定稳定特定反应中间体的条件。 这些测定将与诱变结合使用，以确定控制转运的门 KdpA 并解决它们与 KdpB 中 ATP 驱动的变化耦合的机制。在目标 2 中，我们将 使用单粒子冷冻电镜解析稳定反应中间体的结构，以便可视化 驱动运输的结构要素。在目标 3 中，我们将解决我们意外发现的抑制因子 KdpB 上的磷酸丝氨酸。首要任务是通过以下方式最大限度地降低磷酸化水平： 诱变、磷酸酶处理或生长条件；具有最小磷酸化的活性复合物是 为实现前两个目标所必需的。此外，我们将探索我们的生理作用假设 一旦细胞外 K+ 浓度恢复，丝氨酸磷酸化就会关闭 Kdp 活性。