Potassium transport by the KdpFABC complex

KdpFABC 复合体的钾转运

• 批准号: 10225328
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10225328
• 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.

钾被最早的生物作为细胞质中最普遍的阳离子采用。今天，K+ 跨质膜的梯度在很大程度上是所有细胞的静息潜力，高 细胞质K+浓度对于酶活性，渗透调节和pH稳态至关重要。动物 依靠Na+/K+-ATPase，它是P型ATPase，可在K+中保持约10倍的梯度。而动物 摄入K+丰富的食物并保持细胞外液体，植物，真菌和细菌的体内平衡 在各种环境条件下，可能包括K+中的限制。这些生物已经进化 能够在103到105之间产生梯度的不同K+传输系统。 中等的K+亲和力是组成型表达的，在正常情况下，能够维持 这些梯度。但是，为了在非常低的K+浓度下生存，细菌已经进化了 亲和力，诱导系统，起源于主要的活性转运蛋白。特别是，KDP操纵子是 以微摩尔K+浓度表达，产生一种称为KDPFABC的异常膜复合物 它使用ATP将K+泵入单元。该运输系统代表了前所未有的伙伴关系 在通道状的亚基（KDPA）和泵样亚基（KDPB）之间。前者属于超家族 K+转运蛋白和后者属于P型ATPase家族。作为KDP复合物的一部分 相对于其各自家庭的其他成员，该亚基已被重新利用。特别是，KDPB是 不泵送的P型ATPase，而是使用ATP驱动的构象更改来控制KDPA。 KDPA具有源自K+通道的建筑 化学潜力。我们最近解决了KDPFABC复合物的第一个晶体结构，该结构设置了阶段 为了表征负责此过程的要素，并了解沟通和能量 亚基之间的耦合。基于这种结构，我们开发了特定的假设 通过三个特定目标解决。在AIM 1中，我们将使用生化和生物物理测定法进行 表征反应周期中的步骤，并确定稳定特定反应中间体的条件。 这些测定将与诱变一起使用，以识别控制通过 KDPA并解决将它们与KDPB中ATP驱动的变化耦合的机制。在AIM 2中，我们将 使用单粒子冷冻EM求解稳定反应中间体的结构，以可视化 驱动运输的结构元素。在AIM 3中，我们将解决我们意外的抑制发现 KDPB上的磷serine。首要任务是将磷酸化水平最小化。 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 追求前两个目标所必需的。此外，我们将探讨我们的假设 一旦恢复细胞外K+浓度，丝氨酸磷酸化以关闭KDP活性。

项目成果

Crystal structure of the potassium-importing KdpFABC membrane complex.

• DOI: 10.1038/nature22970
• 发表时间: 2017-06-29
• 期刊: Nature
• 影响因子: 64.8
• 作者: Huang CS;Pedersen BP;Stokes DL
• 通讯作者: Stokes DL

Serine phosphorylation regulates the P-type potassium pump KdpFABC.

• DOI: 10.7554/elife.55480
• 发表时间: 2020-09-21
• 期刊: eLife
• 影响因子: 7.7
• 作者: Sweet ME;Zhang X;Erdjument-Bromage H;Dubey V;Khandelia H;Neubert TA;Pedersen BP;Stokes DL
• 通讯作者: Stokes DL