Serine phosphorylation regulates the P-type potassium pump KdpFABC

Abstract

KdpFABC is an ATP-dependent K+ pump that ensures bacterial survival in K+-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down regulation when K+ levels are restored has not been described. Here we show that KdpFABC is inhibited when cells return to a K+-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K+-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.

Structural basis for Potassium transport by KdpFABC

“…For this work, we introduced the Q116R mutation into KdpA and the S162A mutation into KdpB. As previously reported (Sweet et al, 2020), the resulting protein is fully functional and offers practical advantages for comparative study of reaction intermediates. Whereas the wild-type protein features very high (micromolar) affinity for K + , the Q116R mutation at the mouth of the SF lowers this affinity by three orders of magnitude, and has been used historically for comparison between K + -bound and K + -free states (Epstein et al, 1978; Siebers and Altendorf, 1989).…”

“…Whereas the wild-type protein features very high (micromolar) affinity for K + , the Q116R mutation at the mouth of the SF lowers this affinity by three orders of magnitude, and has been used historically for comparison between K + -bound and K + -free states (Epstein et al, 1978; Siebers and Altendorf, 1989). The S162A mutation prevents phosphorylation of this serine, recently shown to be a regulatory modification that inhibits KdpFABC (Sweet et al, 2020). We expressed this mutant complex in E. coli and purified it in n-decyl-β-D-maltoside detergent using affinity chromatography followed by size-exclusion chromatography (Fig.…”

“…Enzymatic activity of KdpFABC depends on the presence of the transport substrate K + with a K M of 7 mM for the Q116R mutant (Sweet et al, 2020). We therefore compared density maps from samples in the presence and absence of this ion to look for corresponding conformational changes.…”

An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+conditions

“…In both crystal [8] (Figure 1) and cryo-EM structures [9] of the KdpFABC complex, the Serine residue (Ser162) in the TGES motif is found to be phosphorylated [11], while it is well established that the preceding Glutamate residue (Glu161) catalyses the hydrolysis of the phosphorylated aspartate residue. The role of serine phosphorylation has now been traced to the inhibition of the pump when the cells are transferred to a high K + medium.…”

“…The Ser162Ala mutation makes the protein constitutively active, while the phosphorylation mimicking mutation Ser162Asp inactivates the protein [11].…”