Abstract
KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.
Highlights
KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation
Beyond confirming the previously proposed intersubunit tunnel translocation model, our data allow us to propose a molecular mechanism for ion propagation and ATPase coupling, involving residues KdpBF232, KdpBD583, and KdpBK586 (Fig. 6)
In the E1 state, a first K+ ion is drawn past the constriction formed by KdpBF232 to the energy minimum at the proximal binding site (PBS), generated largely by the negatively charged KdpBD583 (Fig. 6a)
Summary
KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. P-type ATPase (KdpB) with a channel-like subunit (KdpA) from the superfamily of K+ transporters (SKT)[4,5] These subunits are augmented by KdpF, a single transmembrane (TM) helix which stabilizes the complex[6], and KdpC, which has been suggested to influence substrate affinity[7], its exact function remains elusive. Its chimeric architecture makes KdpFABC unique among K+ transporters, and ensures the active transport of K+ with an apparent affinity of 2 μM and with high substrate specificity[2,8,9,10] To fulfill this physiological role, both KdpA and KdpB seem to diverge substantially from other members of their families. KdpB is the smallest known P-type ATPase[22], and was long assumed to be unable to transport substrates itself
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