Abstract
KdpFABC is a hetero-tetrameric potassium pump that uses ATP to import potassium and thereby maintain homeostasis in bacteria under stress conditions. KdpA is a channel-like subunit with a selectivity filter that binds potassium from the periplasm. K(+) then moves through a ~40Å-long intramembrane tunnel to reach a canonical binding site in KdpB. KdpB is a P-type ATPase that orchestrates conformational changes associated with the Post-Albers reaction cycle, involving E1 and E2 conformations and formation of an aspartyl phosphate intermediate as a way of coupling ATP hydrolysis to K(+) transport. To elucidate the associated structural changes in a lipid environment, we reconstituted wild-type KdpFABC into lipid nanodiscs and used cryo-EM to image the complex under active turnover. The resulting six high resolution (2.1-2.7 Å) structures provide new insight into the sequence of allosteric changes that produce (1) occlusion of K(+) at the canonical binding site and (2) expulsion of K(+) from this site and into a low-affinity release site. The structures also reveal two types of lipids bound to the complex. Specifically, two structural lipids bind at subunit interfaces and ~20 annular lipids are seen at the periphery of the complex. In addition, we tested functional effects of mutations to residues at the KdpA/KdpB interface. ATPase and transport assays were used to document functional defects that reflect delipidation of structurally compromised complexes. We conclude that lipids play an integral role in structure and function of the KdpFABC complex. SIGNIFICANCE: KdpFABC uses ATP to transport potassium across the plasma membrane of E. coli. To further our understanding of its mechanism, we put purified KdpFABC molecules into membrane bilayers and used cryo-EM to capture structures during active transport. We have thereby produced structures representing all major states of the transport cycle with a high degree of precision. Analysis of these structures reveals new details about two key steps in this cycle and shows lipid molecules bound to the protein. We then introduced mutations at the interface between the two main subunits, which controls passage of potassium across the membrane. Activity measurements reveal how the protein depends on lipid to stabilize the structure and facilitate transport.