Potassium transport by the KdpFABC complex
KdpFABC 复合体的钾转运
基本信息
- 批准号:10225328
- 负责人:
- 金额:$ 34.14万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:ATP HydrolysisATP phosphohydrolaseAddressAdoptedAffectAffinityAnimalsArchitectureBacteriaBindingBinding SitesBiochemicalBiological AssayBiophysicsCationsCell membraneCellsChemicalsCommunicationComplexCoupledCouplingCryoelectron MicroscopyCrystallizationCytoplasmElementsEnvironmentEvolutionExtracellular FluidFamilyFoodGrowthHomeostasisIngestionIonsKineticsLightMass Spectrum AnalysisMeasuresMembraneMembrane PotentialsMolecular ConformationMutagenesisMutationNa(+)-K(+)-Exchanging ATPaseNatureOperonOrganismOsmoregulationPathway interactionsPhosphoric Monoester HydrolasesPhosphorylationPhosphoserinePhylogenetic AnalysisPhysiologicalPlayPotassiumPotassium ChannelProcessPropertyProtonsPumpReactionRestRoleSerineStructureSystemTestingTransmembrane TransportX-Ray Crystallographybaseenzyme activityextracellularfallsinhibitor/antagonistmembermutantnovelpH Homeostasisparticleperiplasmplant fungi
项目摘要
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.
钾被最早的生物体采用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负责所有细胞的静息电位,
细胞质K+浓度对于酶活性、pH调节和pH稳态是必不可少的。动物
依赖于Na+/K+-ATP酶,这是一种P型ATP酶,以维持约10倍的K+梯度。而动物
摄取富含K+的食物并维持细胞外液的稳态,植物、真菌和细菌必须存活
在广泛的环境条件下,其中可能包括K+的限制。这些有机体已经进化出
不同的K+运输系统能够产生103和105之间的梯度。运输工具,
中等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。然而,为了在非常低的K+浓度下生存,细菌进化出了一种高-
作为主要活性转运蛋白发挥功能的亲和诱导系统。特别地,kdp操纵子是
在微摩尔K+浓度下表达,产生称为KdpFABC的异四聚体膜复合物
利用ATP将K+泵入细胞。这一运输系统代表了一种前所未有的伙伴关系
在通道样亚基(KdpA)和泵样亚基(KdpB)之间。前者属于超家族
K ~+转运蛋白属于P型ATP酶家族。作为KDP复合体的一部分,
相对于它们各自家族的其他成员,亚基已经被重新利用。特别是,KdpB是一个
不泵送,而是利用ATP驱动的构象变化来控制KdpA的P型ATP酶。
KdpA具有来源于K+通道的结构,该结构已被调整为使离子对抗电-
化学势我们最近解决了KdpFABC复合物的第一个晶体结构,
描述负责这一过程的元素,并理解通信和能源
子单元之间的耦合。基于这种结构,我们提出了具体的假设,
通过三个具体目标。在目标1中,我们将使用生物化学和生物物理测定,
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。
这些试验将与诱变结合使用,以确定控制转运的门。
KdpA和地址的机制,他们耦合到ATP驱动的KdpB的变化。在目标2中,我们将
使用单粒子cryo-EM来解决稳定的反应中间体的结构,以便可视化
驱动运输的结构要素。在目标3中,我们将讨论我们意想不到的抑制性发现
KdpB上的磷酸丝氨酸。第一个优先事项将是通过以下方式使磷酸化水平最小化:
诱变、磷酸酶处理或生长条件;
为了实现前两个目标。此外,我们还将探讨我们的假设,
一旦细胞外K+浓度恢复,丝氨酸磷酸化关闭Kdp活性。
项目成果
期刊论文数量(5)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Crystal structure of the potassium-importing KdpFABC membrane complex.
- DOI:10.1038/nature22970
- 发表时间:2017-06-29
- 期刊:
- 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:
- 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
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David L. Stokes其他文献
Why conserving species in the wild still matters
- DOI:
10.1007/s10531-018-1509-y - 发表时间:
2018-02-05 - 期刊:
- 影响因子:3.100
- 作者:
David L. Stokes - 通讯作者:
David L. Stokes
Structure of the Calcium Pump from Sarcoplasmic Reticulum at 8 Å Resolution: Architecture of the Transmembrane Helices and Localization of the Binding Site for Thapsigargin
8 Å 分辨率下肌浆网钙泵的结构:跨膜螺旋的结构和毒胡萝卜素结合位点的定位
- DOI:
- 发表时间:
1998 - 期刊:
- 影响因子:2.8
- 作者:
Peijun Zhang;Chikashi Toyoshima;K. Yonekura;G. Inesi;M. Green;David L. Stokes - 通讯作者:
David L. Stokes
Zinc-Induced Conformational Changes in the Cation Diffusion Facilitator YiiP
- DOI:
10.1016/j.bpj.2019.11.2468 - 发表时间:
2020-02-07 - 期刊:
- 影响因子:
- 作者:
Maria L. Lopez;Akiko Koide;Lorena Novoa;Jose M Arguello;Shohei Koide;David L. Stokes - 通讯作者:
David L. Stokes
Mechanism of K<sup>+</sup> transport along the intersubunit tunnel of kdpFABC
- DOI:
10.1016/j.bpj.2022.11.2809 - 发表时间:
2023-02-10 - 期刊:
- 影响因子:
- 作者:
Hridya Valia Madapally;David L. Stokes;Himanshu Khandelia - 通讯作者:
Himanshu Khandelia
Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing.
来自肌浆网的 CaATPase 三维晶体。
- DOI:
- 发表时间:
1990 - 期刊:
- 影响因子:3.4
- 作者:
David L. Stokes;N. Green - 通讯作者:
N. Green
David L. Stokes的其他文献
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{{ truncateString('David L. Stokes', 18)}}的其他基金
Molecular Mechanisms of Ion Transport - Equipment supplement
离子传输的分子机制 - 设备补充
- 批准号:
10798994 - 财政年份:2022
- 资助金额:
$ 34.14万 - 项目类别:
Metal Ion Transport by the Cation Diffusion Facilitator Family
阳离子扩散促进剂家族的金属离子传输
- 批准号:
10083216 - 财政年份:2019
- 资助金额:
$ 34.14万 - 项目类别:
Metal Ion Transport by the Cation Diffusion Facilitator Family
阳离子扩散促进剂家族的金属离子传输
- 批准号:
10592636 - 财政年份:2019
- 资助金额:
$ 34.14万 - 项目类别:
Metal Ion Transport by the Cation Diffusion Facilitator Family
阳离子扩散促进剂家族的金属离子传输
- 批准号:
10319967 - 财政年份:2019
- 资助金额:
$ 34.14万 - 项目类别:
High-throughput Pipeline for Electron Crystallography
电子晶体学高通量管道
- 批准号:
8313999 - 财政年份:2010
- 资助金额:
$ 34.14万 - 项目类别:
TRAINING PROGRAM IN MACROMOLECULAR STRUCTURE AND MECHANISM
大分子结构与机理培训项目
- 批准号:
8291301 - 财政年份:2010
- 资助金额:
$ 34.14万 - 项目类别: