The KdpFABC complex - K+ transport against all odds

Abstract

In bacteria, K⁺ is used to maintain cell volume and osmotic potential. Homeostasis normally involves a network of constitutively expressed transport systems, but in K⁺ deficient environments, the KdpFABC complex uses ATP to pump K⁺ into the cell. This complex appears to be a hybrid of two types of transporters, with KdpA descending from the superfamily of K⁺ transporters and KdpB belonging to the superfamily of P-type ATPases. Studies of enzymatic activity documented a catalytic cycle with hallmarks of classical P-type ATPases and studies of ion transport indicated that K⁺ import into the cytosol occurred in the second half of this cycle in conjunction with hydrolysis of an aspartyl phosphate intermediate. Atomic structures of the KdpFABC complex from X-ray crystallography and cryo-EM have recently revealed conformations before and after formation of this aspartyl phosphate that appear to contradict the functional studies. Specifically, structural comparisons with the archetypal P-type ATPase, SERCA, suggest that K⁺ transport occurs in the first half of the cycle, accompanying formation of the aspartyl phosphate. Further controversy has arisen regarding the path by which K⁺ crosses the membrane. The X-ray structure supports the conventional view that KdpA provides the conduit, whereas cryo-EM structures suggest that K⁺ moves from KdpA through a long, intramembrane tunnel to reach canonical ion binding sites in KdpB from which they are released to the cytosol. This review discusses evidence supporting these contradictory models and identifies key experiments needed to resolve discrepancies and produce a unified model for this fascinating mechanistic hybrid.

Keywords: Active transport; K homeostasis; P-type ATPase; Post-Albers cycle; protein structure; superfamily of K transporters; transport mechanism.

Figure 1:

The Kdp K⁺-uptake system is closely regulated at the transcriptional level. The control is mediated by the sensor kinase KdpD and the response regulator KdpE, which are expressed constitutively. In an environment with K⁺ deficiency, KdpD phosphorylates KdpE, which then promotes transcription of the kdpFABC operon and expression of the KdpFABC complex.

Figure 2:

Generalized Post-Albers cycle for P-type ATPases. E₁ and E₂ represent conformations with ion-binding sites facing the cytoplasm and extracellular medium, respectively. X and Y are ionic species that are transported consecutively out of and into the cytoplasm with stoichiometries of ν and μ, respectively. (X_ν)E₁-P, and E₂(Y_μ) represent occluded states in which the ions are bound within the membrane domain and are unable to exchange with either aqueous phase. E₁P and E₂P represent states in which a conserved catalytic aspartate residue is phosphorylated. In general, formation of this phospho-enzyme accompanies transport of the X ions out of the cytoplasm in the first half cycle, whereas dephosphorylation and transport of the Y ions into the cytoplasm occurs in the second half cycle.

Figure 3:

Monitoring K⁺ transport by the KdpFABC. (A) The florescent dye DiSC3(5) redistributes between the aqueous phase and the inner leaflet of the vesicle membrane as K⁺ transport out of the vesicle generates a negative electric potential. The resulting increase in dye concentration in the inner leaflet causes fluorescence quenching. Accordingly, when ATP was added to a vesicle suspension, K⁺ transport is reflected by decreasing fluorescence as shown in the lower panel. The initial rate of decrease can be used to quantify transport and the exponential shape reflects inhibition of transport as the potential increases. Experimental data were adopted from Damnjanovic et al. (2013). Lipid vesicles are depicted as grey annuli, Kdp as a multicolored complex and DiSC3(5) molecules as 3 rings. (B) Electric currents are generated by the Kdp complex upon release of a small amount of ATP from caged ATP and can be measured with a black lipid membrane setup with proteoliposomes tightly (and thus capacitively) coupled to the black lipid membrane (BLM). The lower panel shows the time course of the electric current detected by electrodes placed on both sides (cis and trans) of the black lipid membrane. The sign of the transient current (which ceases within a few seconds) indicates transport of positive charge out of the liposomes. Experimental data were adopted from Fendler et al. (1996), Copyright 1996 American Chemical Society. I(t) represents a current meter connected to cis and trans electrodes.

Figure 4:

Electrogenic partial reactions of the KdpFABC complex monitored by RH421 fluorescence. (A) Fluorescence decrease induced by the release of ATP from caged ATP in the presence of a saturating concentration of K⁺. The red line represents the fit of an exponential function to the data. (B) The rate constant of the ATP-induced fluorescence decay was independent of the K⁺ concentration, thus indicating that the rate-limiting step occurred before ion binding. (C) The amplitude of the fluorescence decrease was dependent on K⁺ concentration with K_1/2 of 6.8 ± 2.0 μM, which correlates with K_M for the KdpFABC complex. The data were adapted from Damnjanovic and Apell (2014b).

Figure 5:

X-ray crystal structure of the KdpFABC complex in an E₁-like conformation (PDB accession code 5MRW). A: Overview of the structure, which had a resolution of 2.9 Å. The KdpF subunit (cyan) consists in a single membrane spanning helix at the interface of KdpA (green) and KdpB (transmembrane helices brown, P domain blue, A domain yellow, N domain red). KdpC (purple) consists of a single transmembrane helix and a periplasmic domain near the entrance to the selectivity filter of KdpA. B: A cavity (blue surface) runs between the K⁺ ion (purple sphere) in the selectivity filter of KdpA and the water molecule in the canonical ion binding site in KdpB (occupied by a water molecule, red sphere). The cavity has a diameter ≥ 1.4 Å, which is consistent with its occupancy by water.

Figure 6:

Phosphorylation and structural environment of Ser162. This residue is part of the conserved TGES motif in the A domain (yellow), which occupies different configurations in the three structures. (A) Crystal structure which represents the E₁ enzymatic state. (B) State 1 structure from cryo-EM which also represents the E₁ state. (C) State 2 from cryo-EM which represents the E₂ state. The phosphor-serine, S162(P), mediates a salt bridge with two non-conserved residues in the N domain (red) in the X-ray structure. The A domain is known to be highly mobile and although its resolution in cryo-EM maps was not sufficient to see the phosphate, the juxtaposition of A and N domains in these two structures indicated that the salt bridge could not be formed. In state 2, E161 is seen approaching the catalytic aspartate (D307) in the P-domain (blue), which reflects its role in hydrolysis of the phosphoenzyme in the E₂ state.

Figure 7:

Comparison of KdpFABC structures. (A) X-ray crystal structure. (B) State 1 from cryo-EM. (C) State 2 from cryo-EM. Only minor changes were observed in KdpA, KdpC and KdpF. However, the arrangements of N, P and A domains of KdpB have significant differences in E₁-like states and the E₂-P state. The apparent loss of secondary structure in the N-domain from the cryo-EM state 1 is likely to be an artifact of the real-space fitting to the relatively low resolution densities in this region of that map.

Figure 8:

Schematic representation of the two mechanisms proposed for the KdpFABC complex based on structural studies. (A) Mechanism in which K⁺ travels through KdpA and the tunnel serves as a signaling conduit. K⁺ is bound from the periplasm in the selectivity filter of KdpA in the E₁ conformation. A charge migrates through the tunnel and stimulates the ATPase cycle in KdpB. Conformational changes result in movement of the coupling helix (pink) that allows release of K⁺ to the cytoplasm in E₂P. (B) Mechanism in which K⁺ travels from the periplasm to the selectivity filter and through the tunnel from KdpA to KdpB in the E₁ conformation and is then released to the cytosol by KdpB in the P-E₂ conformation.

Figure 9:

Conflicting Post-Albers reaction cycles based on functional and structural analyses of the KdpFABC complex. (A) Mechanism based on functional studies (Siebers and Altendorf, 1989; Damnjanovic and Apell, 2014) that is in agreement with the “classical” cycle for P-type ATPases. In this case, K⁺ is imported in the dephosphorylating half cycle. These studies indicated that protons played an allosteric role during the transport cycle, but did not undergo transport across the membrane. (B) Mechanism based on structural studies (Haupt et al., 2004; Huang et al., 2017; Stock et al., 2018) in which K⁺ is transported in the phosphorylating half cycle.