Potassium transport by the KdpFABC complex

In bacteria there are multiple systems for maintaining potassium homeostasis. TrkH and KtrB are channels that belong to the Superfamily of Potassium Transporters (SKT) and their gating is controled by cytoplasmic domains that belong to the RCK family (TrkA and KtrA, respectively). Under normal growth conditions, bacteria rely on these passive uptake systems, but when K+ levels fall into the micromolar range, a two-component system composed of KdpD/KdpE induces expression of the kdp operon, thus producing the four subunit KdpFABC membrane complex. This complex serves as an ATP-dependent active transporter that actively drives K+ into the cell.
The KdpFABC complex consists of four subunits that work together to couple ATP hydrolysis to the uphill transport of K+ across the membrane. KdpA is a channel-like subunit that belongs to the SKT superfamily. KdpB is a pump-like subunit that belongs to the P-type ATPase superfamily. KdpC and KdpF are single-pass membrane proteins with no obvious homologs outside of the Kdp system.
We were able to crystallize the KdpFABC complex from detergent solution and to solve its structure at 2.9 A resolution using experimental phases based on a Hg derivative. Anomalous signal from SeMet substituted protein helped us build an atomic model into the density. The asymmetric unit consists of three copies of the KdpFABC complex, which each appear to have the same conformation. An unexpected finding was that the protein is phosphorylated on a conserved Ser residue in the A-domain, that normally is involved in dephosphorylation of the aspartyl phosphate at the end of the catalytic cycle. As a result of this phosphorylation, the cytoplasmic domains of KdpB assume a unique juxtaposition not seen in structures of related P-type ATPases. In the overview, the individual subunits are colored the same as the topology diagram above.
KdpA and KdpB have architectures that are consistent with the respective superfamilies. In addition, they have ligands bound at expected sites. In KdpA, there is a K+ ion within the selectivity filter. In KdpB, there is a water molecule at the transmembrane site that is usually used for transport of cations (e.g., Ca2+ in the case of the calcium pump, SERCA).


There is a cavity in the structure that runs ~40 A between these two sites, which we believe will be filled with water and which may provide a means of communication via a proton wire that mediates the mechanistic coupling between the two subunits. In addition, there is a kinked helix in KdpA (pink) which is attached to the cytoplasmic P-domain in KdpB (blue) via a salt bridge. ATP hydrolysis in the P-domain is expected to induce a conformational change that will pull on this kinked helix and thus open a gate to release K+ ions to the cytoplasm. We believe that the periplasmic domain of KdpC (purple) may represent a gating element on the periplasmic side of the membrane.
Based on our structure, we have proposed a mechanistic model for the reaction cycle that pumps K+ across the membrane. This mechanism is based on a Post-Alberts Scheme, which alternates states in which the ion binding site in KdpA is open and closed (open/closed black boxes). The transitions between these states are driven by phosphorylation and dephosphorylation events in the cytosolic domains of KdpB. In the outwards open conformation, K+ binding takes place from the periplasm (1). Binding of potassium leads to charge transfer through the water-filled tunnel from KdpA to the transmembrane domain of KdpB (2). This transfer mimics cation binding to canonical P-type ATPases, which is expected to trigger phosphorylation in KdpB (3). This phosphorylation leads to K+ occlusion in KdpA through a yet undefined element, suggested to be KdpC, forming the potassium occluded E1P state (4). The transition to the E2P state, like in other P-type ATPases, is accompanied by an inclination of the P-domain away from KdpA. This movement pulls the kinked, coupling helix (pink) of KdpA (5), leading to an inwards open E2P state with K+ exchange to the cytosol (6). Dephosphorylation of KdpB returns the cycle back to the open E1 state. The current structure is an inhibited E1 state, shown in blue shade.

Current work is focused on using mutagenesis and functional assays to test these hypotheses.