Potassium transport by the KdpFABC complex

钾被最早的生物体用作细胞质中最普遍的阳离子。今天，K+ 穿过质膜的梯度在很大程度上负责所有细胞的静息电位和高 细胞质中的K+浓度对酶活性、渗透调节和pH动态平衡是必不可少的。动物 依赖于Na+/K+-ATPase，这是一种P型ATPase，使K+保持~10倍的梯度。而动物 摄取富含K+的食物和维持细胞外液的动态平衡，植物、真菌和细菌必须生存 在广泛的环境条件下，可能包括K+的限制。这些生物已经进化成 不同的K+运输系统，能够产生103到105之间的梯度。传送器与 中等的K+亲和力是结构性表达的，在正常情况下，能够保持 这些渐变。然而，为了在很低的K+浓度下生存，细菌进化出了一种高度... 亲和力，可诱导的系统，起主要的主动转运蛋白的作用。特别是，KDP操纵子是 在微摩尔K+浓度下表达，产生一种称为KdpFABC的异四聚体膜复合体 利用三磷酸腺苷将钾离子泵入细胞内。这一运输系统代表了一种前所未有的合作伙伴关系 在类通道亚单位(KdpA)和类泵亚单位(KdpB)之间。前者属于超级大家庭 其中K+转运蛋白属于P型ATPase家族。作为KDP建筑群的一部分，两者 相对于它们各自家族的其他成员，亚单位已经被重新调整了用途。特别是，KdpB是一家 P型ATPase不泵送，而是利用ATP驱动的构象变化来控制KdpA。 KdpA具有从K+通道派生的体系结构，该通道已被适配用于移动离子以对抗电子- 化学势。我们最近解决了KdpFABC络合物的第一个晶体结构，这为 描述负责这一过程的要素，并理解沟通和能量 亚基之间的偶联。基于这一结构，我们制定了具体的假设，这些假设将 通过三个具体目标加以解决。在目标1中，我们将使用生化和生物物理分析来 确定反应周期中的步骤，并确定稳定特定反应中间体的条件。 这些检测将与诱变结合使用，以确定控制运输的门。 KdpA并解决它们与KdpB中由ATP驱动的变化相联系的机制。在目标2中，我们将 用单粒子冷冻-EM方法求解稳定反应中间体的结构 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物 KdpB上的磷酸丝氨酸。当务之急是通过以下方式将磷酸化水平降至最低 诱变、磷酸酶处理或生长条件；具有最小磷酸化的活性络合物 有必要追求前两个目标。此外，我们还将探索我们的假设，以了解 一旦细胞外K+浓度恢复，丝氨酸磷酸化就会关闭KDP的活性。

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.