The bacterial potassium pump Kdp is a unique pump-channel hybrid it is expressed in order to allow cells to grow in low K+ conditions. High levels of
K+ in the cytoplasm are required for cell growth and division as well as for various secondary transport systems.
Kdp is a complex containing 4 subunits and is capable of pumping K+ into the cell against gradients up to 10,000 fold. The largest subunit of the KdpFABC complex, KdpB, resembles a minimal P-type ATPase, whose well-studied members like the Ca-ATPase (SERCA) couple large-scale conformational changes to ATP hydrolysis to ion translocation.
In contrast, the KdpA subunit is related to the Superfamily of K+ transporters (SKT), which includes archetypal K+ channels like KcsA. The historical view is that K+ travels through KdpA, and that ATP-driven conformational changes in KdpB control the gating by an unknown allosteric mechanism. Recent structural advances include our X-ray crystal structure, which provided the first atomic view the complex, and two cryo-EM structures, which suggested a novel pathway for K+. Our X-ray structure revealed a couple of unexpected and unprecedented features.
First, there was a tunnel running between KdpA and KdpB that we proposed was a water-filled conduit for charge transfer between the subunits. Second, a conserved serine on KdpB was phosphorylated and this modification appeared to be inhibitory. Later, two cryo-EM structures revealed a conformational change that supported the idea that after KdpA bound K+ from the periplasm, the ion traveled through the tunnel and exited via KdpB to the cytoplasm. Since these publications, we have been studying effects of serine phosphorylation and solving additional structures by cryo-EM.
We have established physiological conditions that lead to serine phosphorylation which suggest that this modification represents a way to stop Kdp activity once normal levels of K+ are restored to the growth media. We have also used kinetic assays to identify the specific reaction step that is affected.
In addition, we have used cryo-EM to solve several new structures in the presence of specific ligands that capture defined reaction intermediates. The corresponding conformational changes show many of the hallmarks of P-type ATPases. Densities are present within the tunnel and structural analyses suggest that they are most likely water molecules. Although K+ is clearly present in the selectivity filter of KdpA, we are still searching for structural changes that would result in transport to the cytoplasm.