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.
KdpFABC is a membrane complex that allows bacteria to survive in harsh conditions. This project addresses the novel mechanisms by which the parts of this complex collaborate to pump potassium across the cell membrane. More broadly, these studies will contribute to our understanding of the evolution of membrane transport and also will address a novel regulatory mechanism employed by this rescue system.
| Huang, Ching-Shin; Pedersen, Bjørn Panyella; Stokes, David L (2017) Crystal structure of the potassium-importing KdpFABC membrane complex. Nature 546:681-685 |
