Potassium transport systems in bacteria
Organisms from all kingdoms of life maintain elevated levels of potassium in the cytoplasm of their cells. The resulting K+ gradient is used to maintain membrane potential, osmotic pressure and pH. The gradient is used as an energy source for secondary transport systems and is coupled to cell growth and division. In bacteria there are multiple systems for maintaining potassium homeostasis.
Under normal growth conditions, bacteria rely on constitutively expressed uptake systems (Trk and Kup), but when K+ levels fall into the micromolar range, the kdp operon governs expression of the KdpFABC membrane complex. This complex serves as an ATP-dependent transporter that actively drives K+ into the cell.
KdpFABC is a complex comprising a pump and a channel
The KdpFABC complex consists of four subunits that work together to couple ATP hydrolysis to the uphill transport of K+ across the membrane. KdpA is a channel-like subunit that belongs to the Superfamily of K+ Transporters. KdpB is a pump-like subunit that belongs to the P-type ATPase superfamily. KdpC and KdpF are single-pass membrane proteins with no obvious homologs outside of the Kdp system. Like the beta subunits of other P-type ATPases, they most likely serve as structural chaperones to stabilize the complex.
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P-type ATPase Superfamily
This large family includes Ca-ATPase from sarcoplasmic reticulum and plasma membrane, Na/K-ATPase from the plasma membrane of animal cells, H-ATPase from plants and fungi, transition metal pumps prevalent in bacteria, lipid flippases and a new class associated with polyamine transport and ER quality control. KdpFABC is unique in its association with a channel subunit.
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Superfamily of K+ Transporters
This family is characterized by the MPM motif, in which a glycine rich loop constitutes a pore for conduction of K ions. The earliest member of the family - the bacterial channel KcsA - forms a homotetramer, whereas later members evolved after gene duplication generated four copies of this motif. TrkH, KtrB and KdpA are all members of this family.
Architecture of KdpFABC
Our initial structure of the KdpFABC complex was solved by X-ray crystallography. It shows expected architectures for a K+ channel (KdpA, green) and a P-type pump (KdpB), which is characterized by a group of 7 transmembrane helices (brown) and 3 cytoplasmic domains (A-domain in yellow, N-domain in red, P-domain in blue). Surprisingly, this structure revealed a phosphorylated serine residue on the A-domain mediating a salt bridge with the N-domain. Subsequent studies showed that serine phosphorylation represents a regulatory mechanism for inhibiting the pump when K+ is restored to the growth medium such that pumping by KdpFABC is no longer required.
Cryo-EM structures of individual states from the Post-Albers reaction cycle that characterizes P-Type ATPases
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E1 State
Structure in the presence of K+. The absence of ATP or Pi ligands cause the cytoplasmic domains to be disordered, but the overall architecture of KdpB is consistent with the E1 state
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E1 ATP state
Structure in the presence of K+ and the non-hydrolyzable ATP analog, AMP-PCP. Juxtaposition of the cytoplasmic domains is consistent with the E1-P state
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E2-P state
Structure in the presence of BeF3. Position of the A-domain is consistent with the pre-hydrolysis state
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E2 Pi state
Structure in the presence of MgF4. Juxtaposition of the domains is consistent with a post-hydrolysis state
Intra-membrane tunnel
An unprecendented feature of KdpFABC is a 40-A long tunnel connecting the selectivity filter in KdpA with the canonical ion binding site in KdpB. Current models postulate that K+ enters KdpA from the periplasm, moves through this tunnel, and is pumped into the cytoplasm by KdpB. Work is underway to characterize the kinetics and energetics of this process.
Read More
Sweet ME, Larsen C, Zhang X, Schlame M, Pedersen BP, Stokes DL. Structural basis for potassium transport in prokaryotes by KdpFABC. Proc Natl Acad Sci U S A. 2021 Jul 20;118(29). doi: 10.1073/pnas.2105195118.
Dubey V, Stokes DL, Pedersen BP, Khandelia H. An Intracellular Pathway Controlled by the N-terminus of the Pump Subunit Inhibits the Bacterial KdpFABC Ion Pump in High K+ Conditions. J Mol Biol. 2021 Jul 23;433(15):167008. doi: 10.1016/j.jmb.2021.167008.
Sweet ME, Zhang X, Erdjument-Bromage H, Dubey V, Khandelia H, Neubert TA, Pedersen BP, Stokes DL. Serine phosphorylation regulates the P-type potassium pump KdpFABC. Elife. 2020 Sep 21;9. doi: 10.7554/eLife.55480.
Huang CS, Pedersen BP, Stokes DL. Crystal structure of the potassium-importing KdpFABC membrane complex. Nature. 2017 Jun 29;546(7660):681-685. doi: 10.1038/nature22970.