Inhibited KdpFABC transitions into an E1 off-cycle state

Abstract

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC state that we termed E1P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1P states to E2P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such states.

Expulsion mechanism of the substrate-translocating subunit in ECF transporters

“…Our previous work on ECF-FolT2 in lipid nanodiscs provided a starting point for examining asymmetric ~115 kDa ECF transporters at highresolution by cryo-EM 5,22 . We aimed to obtain structures of the complex under turnover conditions, a strategy that was used previously for other transporters [23][24][25] . Accordingly, we incubated ECF-FolT2 with Mg-ATP and folate prior to sample vitrification.…”

Magnesium Transporter MgtA revealed as a Dimeric P-type ATPase

“…25) 63–66 . For the bacterial potassium-importing KdpFABC membrane complex structure, the small protein KdpF has been reported to stabilize the complex 67–69 . Although the ß-subunit of the Na + /K + -pump is 303 aa, it possesses a single pass TM segment that interacts with the larger α-subunit 59,60 .…”

Sensory kinase KdpD is a tandem serine histidine kinase controlling K+pump KdpFABC on the translational and post-transcriptional level

“…The direct phospho-transfer from KdpD to KdpB S162 presented here coincides with multiple previously reported observations: KdpD was shown to interact with KdpB, but for an unknown reason (Babu et al, 2018; Kipschull, 2011). Both KdpFABC and KdpD are also regulated by cardiolipin, suggesting a possible co-localization in the membrane (Schniederberend et al, 2010; Silberberg et al, 2022; Stallkamp et al, 1999). Moreover, the KdpD domain containing the Walker A/B motif is highly conserved among KdpDs from different species, but not found in other sensor kinases (Heermann et al, 2000).…”

“…To prevent this, KdpFABC is inhibited at K + concentrations above 2mM via a phosphorylation of residue S162 of the P-type ATPase KdpB (Huang et al, 2017; Roe et al, 2000; Silberberg et al, 2022; Sweet et al, 2020) (All residue numbers refer to E. coli KdpD and KdpFABC unless otherwise specified). This process appears to be irreversible, and prevents KdpFABC from progressing through its normal catalytic cycle (Silberberg et al, 2022; Sweet et al, 2020). Full inhibition was observed in situ two minutes after exposure to K + (Roe et al, 2000).…”