Potassium transport by the KdpFABC complex

钾被最早的生物体作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。特别地，KdpB是 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 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。

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.