Structural basis for Potassium transport by KdpFABC

Abstract

KdpFABC is an oligomeric K+ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the Superfamily of K+ Transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryo-EM. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC and KdpF remain static. We observe a series of spherical densities that we assign as K+ or water and which define a pathway for K+ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K+ transfer between alternative sites in the membrane domain of KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K+ to the cytoplasm.

Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC

“…Alternatively, these densities could represent waters in the intersubunit tunnel, as proposed recently 11 , although waters are not typically seen in EM at the obtained resolutions 21 .…”

“…Recent structural observations in the E2 state suggest that KdpBK586 pushes the ion into an adjacent low-affinity site, from where it is released through a previously observed inward-open half-channel into the cytosol 11,16 . We tested this hypothesis using MD simulations, and saw that ions were released from the CBS in the E2 state via the proposed half-channel, but did not enter this low-affinity release site (Supplementary Figure 14 c).…”

“…In various cryo-EM structures of KdpFABC, non-protein densities have been observed along the intersubunit tunnel. Although the densities could not be unambiguously assigned, they were suggested to represent K + ions or water molecules 11,15,16 . Due to its higher atomic number, Rb + (Z=37) scatters electrons stronger than K + (Z=19).…”

“…By contrast, KdpBF232A does not increase the resistance to orthovanadate, suggesting that the mutation must affect the catalytic cycle differently. We propose that the increase in ATPase activity observed for KdpFABF232AC is based on the acceleration of the rate-limiting step in KdpFABC turnover, the E1-P/E2-P transition 24 , which is characterized by large rearrangements of the A domain 11,16 . KdpBF232 lies in TM3, which is directly connected to the A domain (Supplementary Figure 12 a).…”

“…KdpA, like other members of the SKT, contains a selectivity filter (SF) that is less conserved than the canonical TVGYG motif of K + channels. This has been proposed to reduce the number of K + binding sites and suggests a lower ion selectivity 10,11 . However, unlike the SKT members TrkH and KtrB, ion transport by KdpFABC was shown to be highly selective for K + over Na + , and ATPase activation by ammonium or Rb + was only possible upon mutation of the KdpA SF region 8,9 .…”