A steady intracellular K⁺ concentration is vital for bacterial cells. Various export and uptake systems jointly regulate bacterial K⁺ homeostasis when facing rapid changes in the environment (Diskowski et al., 2015; Stautz et al., 2021). KdpFABC is a primary active K⁺ uptake system, which is produced when the extracellular K⁺ supply becomes too limited for uptake by less affine translocation systems like KtrAB, TrkAH, or Kup. Due to its high affinity for K⁺ (K_m = 2 µM) and its active transport, KdpFABC can pump K⁺ into the cell even at steep outward-directed gradients of up to 10⁴, thereby guaranteeing a cytosolic K⁺ level of about 200 mM and cell survival (Altendorf et al., 1998; Epstein et al., 1993; Rhoads and Epstein, 1977; Weiden et al., 1967).

The heterotetrametric KdpFABC complex comprises four subunits, namely the channel-like KdpA, a member of the superfamily of K⁺ transporters (SKT) (Durell et al., 2000), the P-type ATPase KdpB (Hesse et al., 1984), the lipid-like stabilizer KdpF (Gassel et al., 1999), and KdpC, whose function is still unknown. KdpB consists of a transmembrane domain (TMD) and the characteristic cytoplasmic nucleotide-binding (N), phosphorylation (P), and actuator (A) domains. Analogous to all P-type ATPases, KdpB follows a Post-Albers reaction scheme, switching between E1 and E2 states that provide alternating access to the substrate-binding site during turnover (Albers, 1967; Huang et al., 2017a; Post et al., 1972; Silberberg et al., 2021; Stock et al., 2018; Sweet et al., 2021). Whilst in its outward-open E1 state, KdpFABC binds ATP in the N domain and takes up K⁺ ions via the selectivity filter in KdpA, which progress into the intersubunit tunnel connecting KdpA and KdpB. Binding of ATP causes rearrangements of the cytoplasmic domains that result in nucleotide coordination between the N and P domains in the E1·ATP state. The γ-phosphate of ATP is coordinated in close proximity to the highly conserved KdpB_D307 of the P domain (all residue numbers refer to Escherichia coli KdpFABC). Upon binding of K⁺ to the canonical substrate-binding site (CBS) of KdpB, KdpB_D307 cleaves off the ATP γ-phosphate via a nucleophilic attack, leading to the autophosphorylation of KdpFABC and progression to the energetically unfavorable E1P state. ATP cleavage releases the N domain from the P domain, allowing relaxing rearrangements of the cytoplasmic domains that convert KdpFABC to the inward-open E2P state, in which K⁺ is released from the CBS to the cytoplasm due to conformational changes in KdpB’s TMD. Finally, KdpB_D307 is dephosphorylated by a water molecule coordinated by KdpB_E161 of the TGES₁₆₂ loop in the A domain, recycling KdpFABC via the non-phosphorylated E2 state back to its E1 ground state (Huang et al., 2017a; Pedersen et al., 2019; Stock et al., 2018; Sweet et al., 2021). Thus, the relative orientation of the three cytoplasmic domains to each other and their nucleotide state (nucleotide-free, nucleotide-bound, or phosphorylated) are crucial for the assignment of catalytic states (Bublitz et al., 2010; Dyla et al., 2020). Notably, while the general catalytic reactions and conformational arrangements of the N, P, and A domains follow the conventional Post-Albers cycle observed for other P-type ATPases, the alternating access of the substrate-binding site in the KdpFABC complex is inverted to accommodate KdpFABC’s unique intersubunit transport mechanism involving KdpA and KdpB (Damnjanovic et al., 2013; Silberberg et al., 2021; Stock et al., 2018).

Being a highly efficient emergency K⁺ uptake system, KdpFABC needs to be tightly regulated in response to changing K⁺ conditions, as both too low and too high potassium concentrations would be toxic (Roe et al., 2000; Stautz et al., 2021). At low K⁺ conditions, transcription of the kdpFABC operon is activated by the K⁺-sensing KdpD/KdpE two-component system (Polarek et al., 1992). Further, post-translational stimulation is conferred by cardiolipin, whose concentration increases as a medium-term response to K⁺ limitation (Schniederberend et al., 2010; Silberberg et al., 2021). Once K⁺ stress has abated ([K⁺_external] > 2 mM), the membrane-embedded KdpFABC is rapidly inhibited to prevent excessive uptake of K⁺ (Roe et al., 2000). This is achieved by a post-translational phosphorylation of KdpB_S162 (yielding KdpFAB_S162-PC), which is part of the highly conserved TGES₁₆₂ motif of the A domain (Huang et al., 2017a; Sweet et al., 2020). In the crystal structure of KdpFABC [5MRW], phosphorylated KdpB_S162 forms salt bridges with KdpB_K357 and KdpB_R363 of the N domain and adopts an unusual E1 conformation. It was suggested that the salt bridge formation inhibits KdpFABC by locking the complex in this state (Huang et al., 2017a). However, the salt bridges were shown to be non-essential to the inhibition mechanism, leaving the role of this conformation unclear (Stock et al., 2018; Sweet et al., 2020). Recent functional studies showed that KdpB_S162 phosphorylation stalls the complex in an intermediate E1P state, preventing the transition to the inward-open E2P state (Sweet et al., 2020).

Here, we set out to address the structural basis for KdpFABC inhibition by KdpB_S162 phosphorylation. The conformational landscape of KdpFABC was probed under different conditions by cryo-EM, yielding 10 structures representative of 6 distinct states that describe the conformational spectrum of the KdpFABC catalytic cycle and resolve the effect of the inhibitory phosphorylation on the conformational plasticity of the complex. Distinct states were further characterized by pulsed electron paramagnetic resonance (EPR) measurements and molecular dynamics (MD) simulations to decipher how KdpB_S162 phosphorylation leads to the inhibition of the complex in the high-energy E1P intermediate.

Previous structural studies of KdpFABC were all conducted in the presence of inhibitors with the aim of stabilizing and obtaining distinct states of the catalytic cycle (Huang et al., 2017a; Silberberg et al., 2021; Stock et al., 2018; Sweet et al., 2021). To describe the structural effects of KdpFABC inhibition by KdpB_S162 phosphorylation, we prepared cryo-EM samples of KdpFABC variants under conditions aimed at covering the conformational landscape of both the non-phosphorylated, active and the phosphorylated, inhibited complex (Table 1; Table 1—source data 1). For this, all KdpFABC variants were produced in Escherichia coli at high K⁺ concentration, which is known to lead to the inhibitory KdpB_S162 phosphorylation (Sweet et al., 2020). KdpFAB_S162AC, a non-phosphorylatable KdpB_S162 variant, and wild-type KdpFABC (KdpFAB_S162-PC) were analyzed under turnover conditions, that is in the presence of saturating KCl and Mg²⁺-ATP concentrations, to gain insights into the dynamic conformational landscape adopted by the complex during catalysis. To supplement these samples, inhibited WT KdpFAB_S162-PC was prepared in the presence of orthovanadate, known to normally arrest P-type ATPases in an E2P or E2·P_i state. Further, the catalytically inactive variant KdpFAB_S162-P/D307NC was prepared under nucleotide-free conditions to investigate E1 apo states. In sum, the 10 maps obtained from the four samples cover the entire KdpFABC conformational cycle, except for the highly transient E1P state after ADP release and the E2 state after dephosphorylation (Table 1—source data 1). All structures exhibit the same intersubunit tunnel described previously, which varies in length depending on the state (Figure 1—figure supplement 1; Huang et al., 2017a; Silberberg et al., 2021; Stock et al., 2018; Sweet et al., 2021). Each tunnel is filled with non-protein densities assigned as potassium ions, which vary slightly in number and position between structures (Figure 1—figure supplement 1B).

Non-inhibited KdpFABC transitions through the Post-Albers cycle under turnover conditions

Previous functional and structural studies have shown that non-phosphorylatable KdpFAB_S162AC can adopt major states of the Post-Albers cycle (Sweet et al., 2021; Sweet et al., 2020). However, the different states were captured with the help of various state-specific inhibitors, limiting our understanding of KdpFABC’s full conformational landscape. To determine the predominant states under turnover conditions, non-phosphorylatable KdpFAB_S162AC was incubated with 2 mM Mg²⁺-ATP and 50 mM KCl for 5 min at 24°C immediately before plunge freezing and analyzed by cryo-EM. From this dataset, we obtained ‘only’ two structures of KdpFABC (Figure 1; Table 1; Figure 1—figure supplements 2 and 3; Table 1—source data 1).

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The first structure was resolved globally at 3.7 Å and derived from ~35% of the initial particle set. The overall orientation of the cytosolic domains is reminiscent of the previously published E1·ATP structures [7NNL], [7NNP], and [7LC3] (root mean square deviations [RMSDs] of cytosolic domains 2.41, 2.61, and 2.69 Å, respectively) (Figure 1A; Figure 1—figure supplement 3E; Table 2; Silberberg et al., 2021; Sweet et al., 2021). However, closer inspection of the N domain reveals no density around the expected γ-phosphate of ATP, whereas additional density is observed near KdpB_D307. We interpret that ADP, instead of ATP, is bound and the catalytic aspartate KdpB_D307 has been phosphorylated (Figure 1B). An additional density observed near the phosphorylated KdpB_D307 coincides with a Mg²⁺ ion in the E1P·ADP SERCA structure [1T5T] (Sørensen et al., 2004), and has thus likewise been assigned as a coordinating Mg²⁺ ion. Altogether, this conformation represents an E1P·ADP state, following phosphorylation from ATP but preceding the release of ADP. The second structure obtained from the KdpFAB_S162AC sample under turnover conditions was resolved globally at 4.0 Å from ~28% of the initial particle set. In the respective cryo-EM map, a density is observable immediately adjacent to KdpB_D307 and the TGES₁₆₂ loop is in close proximity, which is the hallmark of an E2P or E2·P_i state (Figure 1C, D). The overall orientation of the cytosolic domains more closely resembles that of the E2·P_i structures (RMSD of 2.66 and 2.4 Å for [7BH2] and [7BGY]) than that of the E2P structure (RMSDs of 11 Å for [7LC6]) (Figure 1—figure supplement 3J; Table 2; Sweet et al., 2021). However, on closer inspection, the density fits better to a to a covalently bound phosphate than to a coordinated P_i (Figure 1D). In light of the ambiguity at the given resolution, we have assigned this structure to an E2P state which might be transitioning to an E2·P_i. In comparison to the E1P·ADP state, the A domain in the E2P state has undergone a tilt of 60° and a rotation of 64° around the P domain, positioning the TGES₁₆₂ loop to dephosphorylate KdpB_D307 in the P domain, while the P domain is also tilted by 40° (Figure 1—figure supplement 4).

Table 2

State		E1 apo open				E1·ATP				E1P·ADP		E2P	E2P/E2·Pi	E2·Pi			E1 apo tight	E1P tight		Crystal Structure
	PDB	6HRA	7BH1	7ZRI	7ZRJ	7LC3	7NNL	7NNP	7ZRG	7ZRM	7ZRK	7LC6	7ZRL	6HRB	7BGY	7BH2	7ZRH	7ZRE	7ZRD	5MRW A	5MRW B
E1 apo open	6HRA (3.7 Å)		9.67	8.28	4.14	10.31	9.69	10.02	8.59	9.32	9.50	17.15	25.42	24.91	25.01	25.53	14.25	13.25	13.07	14.29	12.06
	7BH1 (3.4 Å)	9.67		5.65	8.16	15.66	15.06	15.39	10.40	14.43	14.79	22.25	30.81	30.36	30.40	31.00	14.78	14.84	14.67	18.00	15.26
	7ZRI (3.5 Å)	8.28	5.65		6.36	14.83	14.14	14.47	9.61	13.49	13.86	22.58	31.33	30.85	30.87	31.43	15.11	15.64	15.52	18.50	15.58
	7ZRJ (3.7 Å)	4.14	8.16	6.36		12.27	11.57	11.91	8.93	11.06	11.34	18.65	27.15	26.67	26.72	27.23	14.99	14.41	14.23	16.04	13.54
E1·ATP	7LC3 (3.2 Å)	10.31	15.66	14.83	12.27		2.10	1.96	7.50	2.69	2.44	17.84	25.08	24.65	24.63	25.37	18.67	17.54	17.29	17.30	15.01
	7NNL (3.1 Å)	9.69	15.06	14.14	11.57	2.10		1.75	6.86	2.41	2.17	17.95	25.36	24.92	24.91	25.62	18.28	17.25	17.00	17.19	14.85
	7NNP (3.2 Å)	10.02	15.39	14.47	11.91	1.96	1.75		7.23	2.61	2.34	17.84	25.16	24.72	24.70	25.42	18.42	17.34	17.11	17.14	14.81
	7ZRG (3.5 Å)	8.59	10.40	9.61	8.93	7.50	6.86	7.23		6.13	6.57	20.08	28.13	27.69	27.68	28.40	16.59	16.21	16.01	17.80	15.33
E1P·ADP	7ZRM (3.7 Å)	9.32	14.43	13.49	11.06	2.69	2.41	2.61	6.13		2.12	18.25	25.75	25.32	25.32	26.03	18.00	17.05	16.81	17.25	14.91
	7ZRK (3.1 Å)	9.50	14.79	13.86	11.34	2.44	2.17	2.34	6.57	2.12		17.84	25.32	24.90	24.88	25.59	18.23	17.20	16.96	17.24	14.86
E2P	7LC6 (3.7 Å)	17.15	22.25	22.58	18.65	17.84	17.95	17.84	20.08	18.25	17.84		11.03	10.44	10.32	10.71	24.86	22.44	22.32	19.25	19.44
E2P/E2·Pi	7ZRL (4.0 Å)	25.42	30.81	31.33	27.15	25.08	25.36	25.16	28.13	25.75	25.32	11.03		2.52	2.43	2.66	30.42	27.40	27.30	22.80	23.23
E2·Pi	6HRB (4.0 Å)	24.91	30.36	30.85	26.67	24.65	24.92	24.72	27.69	25.32	24.90	10.44	2.52		1.98	2.18	30.05	27.05	26.96	22.42	22.93
	7BGY (2.9 Å)	25.01	30.40	30.87	26.72	24.63	24.91	24.70	27.68	25.32	24.88	10.32	2.43	1.98		1.74	30.18	27.20	27.10	22.61	22.37
	7BH2 (3.0 Å)	25.53	31.00	31.43	27.23	25.37	25.62	25.42	28.40	26.03	25.59	10.71	2.66	2.18	1.74		30.76	27.77	27.68	23.11	22.86
E1 apo tight	7ZRH (3.4 Å)	14.25	14.78	15.11	14.99	18.67	18.28	18.42	16.59	18.00	18.23	24.86	30.42	30.05	30.18	30.76		4.84	5.01	9.85	7.53
E1P tight	7ZRE (3.4 Å)	13.25	14.84	15.64	14.41	17.54	17.25	17.34	16.21	17.05	17.20	22.44	27.40	27.05	27.20	27.77	4.84		1.59	6.50	4.92
	7ZRD (3.7 Å)	13.07	14.67	15.52	14.23	17.29	17.00	17.11	16.01	16.81	16.96	22.32	27.30	26.96	27.10	27.68	5.01	1.59		6.51	4.87
Crystal Structure	5MRW A (2.9 Å)	14.29	18.00	18.50	16.04	17.30	17.19	17.14	17.80	17.25	17.24	19.25	22.80	22.42	22.61	23.11	9.85	6.50	6.51
	5MRW B (2.9 Å)	12.06	15.26	15.58	13.54	15.01	14.85	14.81	15.33	14.91	14.86	19.44	23.23	22.93	22.37	22.86	7.53	4.92	4.87

The E1P·ADP and E2P structures obtained for KdpFAB_S162AC confirm that, in the absence of the inhibitory KdpB_S162 phosphorylation, KdpFABC progresses through the entire Post-Albers cycle under turnover conditions, with the ADP release in the E1 state and the orthophosphate release in the E2 state likely being the rate-limiting steps that lead to an accumulation of the observed states.

Inhibited E1P KdpFABC adopts an off-cycle state

To study the structural implications of KdpFABC inhibition by KdpB_S162 phosphorylation, we next analyzed the conformational landscape of WT KdpFAB_S162-PC under turnover conditions. To our surprise, the dataset disclosed a higher degree of conformational variability than KdpFAB_S162AC, yielding three distinct cryo-EM structures (Figure 2; Table 1; Figure 2—figure supplements 1 and 2; Table 1—source data 1). The obtained structures show significant deviations in the cytoplasmic region of KdpB, indicative of different positions in the Post-Albers cycle.

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The first KdpFAB_S162-PC turnover structure, resolved at an overall resolution of 3.5 Å and derived from ~9% of the initial particle set, roughly resembles the E1·ATP structures [7NNL], [7NNP], and [7LC3] (RMSDs of cytosolic domains 6.86, 7.23, and 7.50 Å, respectively) (Silberberg et al., 2021; Figure 2A; Figure 2—figure supplement 2E; Table 2). However, a closer analysis of the cytosolic domains and the bound nucleotide reveals significant differences. While ATP is coordinated in a similar fashion as previously observed (Silberberg et al., 2021; Sweet et al., 2021), the N domain is slightly displaced relative to the P domain, providing more access to the nucleotide-binding site (Figure 2A, B; Figure 2—figure supplement 2E). We interpret this structure as an E1·ATP state at an early stage of nucleotide binding and refer to it as E1·ATP_early. By contrast, inhibitors such as AMPPCP likely stabilize the latest possible and otherwise transient E1·ATP state immediately before phosphorylation, explaining the discrepancy between the AMPPCP-stabilized [7NNL] and the E1·ATP state obtained here under turnover conditions. Alternatively, this structure could represent an artefactual ‘ADP-inhibited’ state, in which ADP binds to the N domain and prevents ATP binding. While this is supported by the relatively weak density of the γ-phosphate and the more open conformation, it would be surprising, considering the high excess of ATP used during sample preparation. The second KdpFAB_S162-PC turnover structure, resolved at an overall resolution of 3.1 Å and representing ~31% of the initial particle set, is very similar to the above-described E1P·ADP state of KdpFAB_S162AC (RMSD of cytosolic domains 2.12 Å) (Figure 2C, D; Figure 2—figure supplement 2J; Table 2).

While the first two structures represent known states of the catalytic cycle, the third KdpFAB_S162-PC structure obtained under turnover conditions shows an unusual compact conformation of the cytosolic domains not yet observed in the Post-Albers cycle of other P-type ATPases (Figure 2E, F). The structure was resolved at an overall resolution of 3.4 Å and derived from ~14% of the initial particle set. Strikingly, the N domain is closely associated with the A domain, thereby disrupting the nucleotide-binding site between the N and P domains (Figure 2E, F; Figure 2—figure supplement 3). Closer inspection of the nucleotide-binding site shows that the catalytic aspartate KdpB_D307 is phosphorylated, but not located in proximity to the TGES₁₆₂ loop of the A domain (Figure 2F). This indicates that the state is adopted after the canonical E1P state, which, in a normal, non-inhibited catalytic cycle, would transition into an E2P state. Due to its compact organization, we termed this state E1P tight.

Previous biochemical studies have shown that KdpB_S162 phosphorylation inhibits KdpFABC by preventing the transition to an E2 state and stalling it in an E1P state (Sweet et al., 2020). In line with these observations, we could not identify any state following E1P in the Post-Albers cycle for KdpFAB_S162-PC, despite the turnover conditions used. Based on this, we hypothesized that the novel E1P tight state observed represents a non-Post-Albers state that is adopted because KdpFAB_S162-PC cannot proceed to the E2P state. To further put this to test, we analyzed the conformations adopted by WT KdpFAB_S162-PC in the presence of the phosphate mimic orthovanadate, which has been shown to trap P-type ATPases in an E2P state and, of all E2 state inhibitors, best mimics the charge distribution of a bound phosphate (Table 1; Figure 2—figure supplements 4 and 5; Table 1—source data 1; Clausen et al., 2016; Pedersen et al., 2019). Interestingly, KdpFAB_S162-PC incubated with 2 mM orthovanadate prior to cryo-EM sample preparation did not conform to this behavior. Instead, the major fraction of this sample (~62% of the initial particle set) resulted in an E1P tight state, resolved at 3.3 Å, which is shows no large differences to the E1P tight state obtained under turnover conditions (RMSD of cytosolic domains 1.59 Å) (Figure 2—figure supplement 4H; Table 2). The position of the orthovanadate coincides with that adopted by the phosphorylated KdpB_D307, verifying the assignment of this state as a phosphorylated E1P intermediate (Figure 2—figure supplement 5). Only a minor fraction of the orthovanadate-stabilized sample (~11% of the initial particle set) adopted an E2P state. Despite the rather poor resolution of 7.4 Å, the catalytic state could be confirmed by comparison of the cryo-EM map with the previously published E2·P_i structure [7BH2] (Figure 2—figure supplement 4I; Sweet et al., 2021). The minor fraction observed in an E2 state likely represents residual KdpFABC lacking KdpB_S162 phosphorylation, as it is in good agreement with the residual ATPase activity level found in the sample (Figure 2—figure supplement 5C; Sweet et al., 2020). The fact that, in the presence of orthovanadate, KdpFAB_S162-PC adopts the E1P tight state instead of an E2P state strongly supports the idea that the inhibited complex cannot transition into an E2 state but instead adopts an off-cycle E1P state after ADP dissociation.

A tight state is also formed in the absence of nucleotide

Interestingly, the close association of the N domain with the A domain in the E1P tight conformation also shows some similarities to the E1 crystal structure of KdpFABC [5MRW], the first structure of KdpFABC ever solved (RMSD of cytosolic domains 4.92 Å) (Table 2; Huang et al., 2017a). Unlike our E1P tight state, the crystal structure is nucleotide-free and does not contain a phosphorylated KdpB_D307, raising the question how it fits in the conformational landscape of KdpFABC. To further investigate the role of the tight state in the conformational cycle of KdpFABC, we prepared a cryo-EM sample of KdpFAB_S162-P/D307NC under nucleotide-free conditions. This mutant is catalytically inactive and thus restricted to E1 states preceding phosphorylation in the P domain. In total, we were able to obtain three distinct structures from this preparation, which we assigned to two states relevant to the transport cycle (Figure 3, Table 1; Figure 3—figure supplements 1 and 2; Table 1—source data 1).

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The first state, composed of 26% of the initial particle set from the nucleotide-free sample, corresponds to the canonical Post-Albers E1 state that precedes ATP binding and resembles the E1 states [6HRA] and [7BH1] reported previously (Stock et al., 2018; Sweet et al., 2021; Figure 3A; Figure 3—figure supplement 2E). We have termed this state the E1 apo open state, as the N and P domains are far apart to provide access to the nucleotide-binding site. The cytosolic domains of KdpB reveal a high conformational heterogeneity, evidenced by their lower local resolution (Figure 3—figure supplement 2B, G). Focused 3D classification allowed the distinction of two substates, resolved globally at 3.5 and 3.7 Å, differing in the relative position of the N to the P domain (RMSDs between cytosolic domains of 8.28 and 5.65 Å for substate 1 to [6HRA] and [7BH1], respectively, and 4.14 and 8.16 Å for substate 2 to [6HRA] and [7BH1], respectively, with an RMSD of 6.36 Å between substates 1 and 2) (Figure 3—figure supplement 2J; Table 2). The high degree of flexibility of the N domain in these open states could facilitate nucleotide binding at the start of the Post-Albers cycle. However, it remains unknown whether KdpB can already bind ATP to its N domain in the preceding E2 states, as was shown for other P-type ATPases, which would call the physiological relevance of the E1 apo states into question (Jensen et al., 2006).

The second state, featuring one structure resolved globally at 3.4 Å and represented by 19% of the initial particle set, shows a compact conformation of the cytosolic domains with the N and A domains in close contact, providing no space for a nucleotide to bind (Figure 3B). This state is similar, but not identical to the one observed in the E1P tight state and [5MRW] (RMSDs of cytosolic domains 4.84 and 7.53 Å, respectively), and we have termed it the E1 apo tight state (Figure 3—figure supplement 2O; Table 2). The presence of a similar tight conformation in both the E1P and the E1 apo states of KdpB_S162-P shows that, while the inhibitory KdpB_S162 phosphorylation appears to be a prerequisite, the observed close association of the N and A domains can occur before or after the binding and cleavage of ATP.

The tight state interaction between the N and A domains is itself not the cause of inhibition

Structures with a tight arrangement of the A and N domains were only obtained in cryo-EM samples featuring KdpB_S162-P. To evaluate the dependency of tight state formation on KdpB_S162 phosphorylation, we assessed the full conformational flexibility of the N domain using pulsed EPR spectroscopy. Distances between the N and P domains were measured with the labeled residues KdpB_A407CR1 and KdpB_A494CR1 (Figure 4A, B; Figure 4—figure supplement 1). Variants were produced at high K⁺ concentrations to confer KdpB_S162 phosphorylation. EPR analysis of KdpFAB_S162-P/D307NC in the absence of nucleotide resulted in distances that resemble the E1 apo tight (34%) and the E1 apo open (66%) states (Figure 4B). This corroborates the cryo-EM results that both tight and open states are adopted in the same sample at nucleotide-free conditions. In contrast, the non-phosphorylatable variant KdpB_S162A resulted in a distance distribution showing a significant decrease of the E1 apo tight state (18%). This strongly indicates that the tight state is stabilized by KdpB_S162 phosphorylation, although it still exists to a small extent even in the absence of the inhibitory phosphorylation.

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To inspect what makes the off-cycle tight states energetically preferable, we quantified the cytosolic domain interactions of all E1 states obtained in this study using contact analysis by MD simulation (Figure 4C; Figure 4—figure supplement 2). While no large differences are observed for the contacts between A and P or N and P domains, the off-cycle E1 tight states (E1 apo tight, E1P tight) are the only ones to show a higher number of interactions between the A and N domains. In fact, they share a nearly identical number of contacts, indicating that the A and N domains move in a concerted manner, similar to previous observations by MD simulations (Dubey et al., 2021). The most evident interaction seen in all three tight states is formed by the salt bridges between the phosphate of KdpB_S162-P in the A domain and KdpB_K357/R363 in the N domain, which were first described in [5MRW] (Huang et al., 2017a). However, functional studies have shown that these salt bridges are not essential for the inhibition (Sweet et al., 2020), and neither are they sufficient to fully arrest a tight state, as shown here. In agreement with this, EPR measurements with KdpFAB_S162-P/D307NC in the presence of the ATP analogue AMPPCP result in a distance distribution that shows a single and well-defined distance, which corresponds to the E1·ATP state. Hence, the salt bridges and the enhanced interaction platform between the A and N domain seen in the tight states likely have a stabilizing role, but can be easily broken and are themselves not the main cause of inhibition.

The E1P tight state is the consequence of an impaired E1P/E2P transition

As the compact arrangement found in the E1 tight states itself is not the determining factor of inhibition, the question remains how K⁺ transport is blocked in KdpFAB_S162-PC and what role the E1P tight state plays. In a non-inhibited transport cycle, KdpFABC rapidly relaxes from the high-energy E1P state into the E2P state. To identify what structural determinants enable the arrest before this transition, we compared the E1P tight structure with the other structures obtained in this study (Figure 5). The E1P tight state features a tilt of the A domain by 26° in helix 4 (KdpB_198–208) that is not found in the other E1 states, including the E1 apo tight state (Figure 5B, C – arrow 1). This tilt is reminiscent of the movement the A domain undergoes during the E1P/E2P transition (60° tilt, Figure 1—figure supplement 4), but it is not as far and does not feature the rotation around the P domain (Figure 5—figure supplement 1A–C). Moreover, the P domain does not show the tilt observed in the normal E1P/E2P transition (Figure 5—figure supplement 1D). Notably, these are the movements that bring KdpB_D307-P in close proximity of KdpB_S162 in the catalytic cycle (Figure 5—figure supplement 2, Video 1). In an inhibited state, where both side chains are phosphorylated, such a transition is most likely impaired due to the large charge repulsion. Moreover, a comparison of the E1P tight structure with the E1P·ADP structure, its most immediate precursor in the conformational cycle obtained, reveals a number of significant rearrangements within the P domain (Figure 5B, C). First, Helix 6 (KdpB_538–545) is partially unwound and has moved away from helix 5 toward the A domain, alongside the tilting of helix 4 of the A domain (Figure 5B, C – arrow 2). Second, and of particular interest, are the additional local changes that occur in the immediate vicinity of the phosphorylated KdpB_D307. In the E1P·ADP structure, the catalytic aspartyl phosphate, located in the D₃₀₇KTG signature motif, points toward the negatively charged KdpB_D518/D522. This strain is likely to become even more unfavorable once ADP dissociates in the E1P state, as the Mg²⁺ associated with the ADP partially shields these clashes. The ensuing repulsion might serve as a driving force for the system to relax into the E2 state in the catalytic cycle. By contrast, the D₃₀₇KTG loop is largely uncoiled in the E1P tight state, with the phosphorylated KdpB_D307 pointing in the opposite direction, releasing this electrostatic strain (Figure 5C – arrow 4). This orientation is further stabilized by a salt bridge formed with KdpB_K499. The uncoiling in the E1P tight state is likely mediated by the movement of the N domain toward the A domain, as the N domain is directly connected to the D₃₀₇KTG loop (Figure 5C – arrow 3). Altogether, we propose that, in the presence of the inhibitory KdpB_S162-P, the high-energy E1P·ADP state can no longer transition into an E2P state after release of ADP and Mg²⁺. As a consequence, the conformational changes observed in the E1P tight state likely ease the electrostatic tensions of the phosphorylated P domain and stall the system in a ‘relaxed’ off-cycle state.

Figure 5 with 2 supplements see all

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Video 1

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We set out to deepen our understanding of the structural basis of KdpFABC inhibition via KdpB_S162 phosphorylation by sampling the conformational landscape under various conditions by cryo-EM. The 10 cryo-EM maps of KdpFABC, which represent six distinct catalytic states, cover close to the full conformational landscape of KdpFABC and, most importantly, uncover a non-Post-Albers regulatory off-cycle state involved in KdpFABC inhibition (Figure 6).

Figure 6

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The catalytic states identified here closely adhere to the classical Post-Albers cycle of KdpFABC (Video 1). In the outward-facing E1 apo open state, the nucleotide-binding domain is widely accessible, and the N domain shows a high degree of flexibility, which likely enhances the efficiency of nucleotide binding from the environment. Once a nucleotide is bound, the N domain reorients toward the P domain for shared coordination of the nucleotide in the E1·ATP_early state (Figure 6, transition 1a). This conformation shows a slightly more open nucleotide-bound state than the previously reported AMPPCP-stabilized E1·ATP state, which likely can progress closer to ATP cleavage (Silberberg et al., 2021; Sweet et al., 2021). Subsequently, ATP cleavage leads to the phosphorylation of the catalytic KdpB_D307 in the P domain, forming the E1P·ADP intermediate (Figure 6, transition 2), which could be structurally isolated in this study under turnover conditions. The accumulation of the E1P·ADP state in both turnover samples analyzed in this study suggests that ADP and Mg²⁺ release (Figure 6, transition 3) is the rate-limiting step of KdpFABC turnover. Following the catalytic cycle, the high-energy E1P state progresses to the E2P state, whereby large rearrangements bring the conserved TGES₁₆₂ loop in the A domain near the catalytic site of the P domain. This transition is accompanied by conformational changes in the TMD of KdpB, which switches the complex from an outward- to an inward-open state with respect to the CBS (Figure 6, transition 4a). K⁺ is released to the cytosol and KdpB_D307-P is dephosphorylated by the TGES₁₆₂ loop (Figure 6, transition 5). Subsequently, the complex cycles back to the E1 apo open state (Figure 6, transition 6).

Whereas the structures obtained for KdpFAB_S162-PC in the E1 apo open, E1·ATP, E1P·ADP, and E2P states align well with corresponding states in the Post-Albers cycle of other P-type ATPases, the nucleotide-free E1 apo tight and E1P tight states do not. Their overall compact fold is facilitated by KdpB_S162-P, which increases the contacts of the N and A domains. While the compact fold itself is not the main cause of stalling KdpFABC, it might contribute to stabilizing the inhibited complex limiting the innate flexibility of the N domain. Notably, the E1 apo tight state likely has no major physiological relevance, as in the presence of ATP, it would progress through the catalytic cycle up to the point of inhibition.

By contrast, we propose that the E1P tight state is involved in stalling the KdpFABC complex. We suggest that this state represents the biochemically described E1P inhibited state (Sweet et al., 2020), and is adopted after ADP release from the high-energy E1P state, when KdpFABC attempts to relax into the E2P state (Figure 6, transition 4b). This attempt would explain the partial tilt of the A domain in helix 4, observed only in the E1P tight structure. As suggested before (Sweet et al., 2020), the full relaxation to the E2P state is however hindered in the inhibited KdpFAB_S162-PC by a repulsion between the catalytic phosphate in the P domain (KdpB_D307-P) and the inhibitory phosphate in the A domain (KdpB_S162-P), which would come in close proximity during the transition from E1P to E2P (Figure 6, transition 4a, Video 1). The adopted stalled E1P tight state is likely an energetically favored state between E1P and E2P, easing the high-energy constraints around the phosphorylated KdpB_D307. In good agreement with this hypothesis, KdpFAB_S162-PC was stabilized in the E1P tight state even in the presence of the inhibitor orthovanadate, known to otherwise stabilize an E2P state. Notably, orthovanadate has the same charge distribution as the E1P tight state with phosphorylated KdpB_D307. By contrast, KdpFAB_S162-PC in the presence of AlF₄⁻, which has a lower negative charge, could previously be stabilized in an E2P state (Stock et al., 2018), likely because the electrostatic repulsion is lower, making the E1P/E2P transition more favorable.

For a long time, E1P states of P-type ATPases have been biochemically characterized as high-energy intermediates. However, the structural determinants for this energetic unfavorability have yet to be described. The conformational changes we observe between the E1P·ADP state and the relaxed E1P tight state offer a first clue as to the regions involved in destabilizing the E1P state and triggering the transition to the E2P state. In the E1P tight state, the electrostatic tensions of the E1P·ADP state are eased by rearrangements of the catalytic aspartate and the surrounding side chains, likely supported by a pulling of the N domain on the D₃₀₇KTG loop as it associates with the A domain during the E1P to E1P tight transition. This could be the main stabilizing role of the tight state in KdpB_S162-P inhibition. Further, the structural differences in the E1P tight state also involve a movement of helix 6 in the P domain, which is not in the immediate proximity of the catalytic KdpB_D307 phosphate. This helix is in close proximity to the A domain and next one of the two connections of the P domain to the TMD, which undergo large conformational changes during the E1/E2 transition. Thus, it may be an important element in signaling the ATPase to initiate the transition to E2P in the TMD from an outward- to an inward-facing state.

The regulation by inactive states outside the Post-Albers cycle has been postulated for other P-type ATPases (Dyla et al., 2020). Observation of translocation by the H⁺ ATPase AHA2 on a single-molecule level revealed that the pump stochastically enters inactive states, from which it can return to its active form spontaneously (Veshaguri et al., 2016). These states are adopted from the E1 state, similar to the E1 apo tight state observed under nucleotide-free conditions for KdpFABC. However, no full inhibition in the E1P state was observed for AHA2, indicating a different mechanism. A crystal structure of the Ca²⁺ pump SERCA was also proposed to represent a non-Post-Albers state (Dyla et al., 2020; Toyoshima et al., 2000). Yet, the organization of the cytosolic domains differs significantly from the tight state seen for KdpB, and a physiological role for this structure remains unclear. Altogether, the structural basis for KdpFABC inhibition by KdpB_S162 phosphorylation presented here describes in detail the involvement of a non-Post-Albers state with a clear regulatory role in P-type ATPase turnover. It extends the conformational landscape and strongly supports the emerging idea of non-catalytic off-cycle states with important physiological roles.

Key materials and software used in this work are listed in a key resources table.

The three-dimensional cryo-EM densities and corresponding modeled coordinates generated have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank under the accession numbers summarized in Table 4. The depositions include maps calculated with higher b-factors, both half-maps and the mask used for the final FSC calculation.

1. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zrg. KdpFABC WT (KdpB-Ser162-P) in an E1·ATPearly state under turnover conditions.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zrg
2. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zrk. KdpFABC WT (KdpB-Ser162-P) in an E1P·ADP state under turnover conditions.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zrk
3. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zre. KdpFABC WT (KdpB-Ser162-P) in an E1P-tight state under turnover conditions.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zre
4. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zrd. KdpFABC WT (KdpB-Ser162-P) in an E1P-tight state in presence of orthovanadate.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zrd
5. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zrh. KdpFAB(Ser162-P,D307N)C in an E1 apo tight state.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zrh
6. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zri. KdpFAB(Ser162-P,D307N)C in an E1 apo open 1 state.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zri
7. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zrj. KdpFAB(Ser162-P,D307N)C in an E1 apo open 2 state.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zrj
8. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zrm. KdpFAB(S162A) in an E1P·ADP state under turnover conditions.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zrm
9. 1. Silberberg JM

   (2022) Electron Microscopy Data Bank

   ID 7zrl. KdpFABC WT (KdpB-Ser162-P) in an E2P state under turnover conditions.

   https://www.ebi.ac.uk/pdbe/entry/pdb/7zrl
10. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14347. KdpFABC WT (KdpB-Ser162-P) in an E2P state in presence of orthovanadate.

    https://www.ebi.ac.uk/emdb/EMD-14347
11. 1. Silberberg JM

    (2022) Electron Microscopy Public Image Archive

    ID EMPIAR-11232. Single particle cryo-EM of KdpFABC WT (KdpB-Ser162-P) under turnover condition.

    https://www.ebi.ac.uk/empiar/EMPIAR-11232/
12. 1. Silberberg JM

    (2022) Electron Microscopy Public Image Archive

    ID EMPIAR-11231. Single particle cryo-EM of KdpFAB(S162A)C under turnover condition.

    https://www.ebi.ac.uk/empiar/EMPIAR-11231/
13. 1. Silberberg JM

    (2022) Electron Microscopy Public Image Archive

    ID EMPIAR-11230. Single particle cryo-EM of KdpFABC WT (KDpB-Ser162-P) in presence of orthovanadate.

    https://www.ebi.ac.uk/empiar/EMPIAR-11230/
14. 1. Silberberg JM

    (2022) Electron Microscopy Public Image Archive

    ID EMPIAR-11229. Single particle cryo-EM of KdpFAB(Ser162-P, D307N)C in apo condition.

    https://www.ebi.ac.uk/empiar/EMPIAR-11229/
15. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMDB-14913. KdpFABC WT (KdpB-Ser162-P) in an E1·ATPearly state under turnover conditions.

    https://www.ebi.ac.uk/emdb/EMD-14913
16. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14917. KdpFABC WT (KdpB-Ser162-P) in an E1P·ADP state under turnover conditions.

    https://www.ebi.ac.uk/emdb/EMD-14917
17. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14912. KdpFABC WT (KdpB-Ser162-P) in an E1P-tight state under turnover conditions.

    https://www.ebi.ac.uk/emdb/EMD-14912
18. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14911. KdpFABC WT (KdpB-Ser162-P) in an E1P-tight state in presence of orthovanadate.

    https://www.ebi.ac.uk/emdb/EMD-14911
19. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14914. KdpFAB(Ser162-P,D307N)C in an E1 apo tight state.

    https://www.ebi.ac.uk/emdb/EMD-14914
20. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14915. KdpFAB(Ser162-P,D307N)C in an E1 apo open 1 state.

    https://www.ebi.ac.uk/emdb/EMD-14915
21. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14916. KdpFAB(Ser162-P,D307N)C in an E1 apo open 2 state.

    https://www.ebi.ac.uk/emdb/EMD-14916
22. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14919. KdpFAB(S162A) in an E1P·ADP state under turnover conditions.

    https://www.ebi.ac.uk/emdb/EMD-14919
23. 1. Silberberg JM

    (2022) Electron Microscopy Data Bank

    ID EMD-14918. KdpFABC WT (KdpB-Ser162-P) in an E2P state under turnover conditions.

    https://www.ebi.ac.uk/emdb/EMD-14918

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Author details

1. Jakob M Silberberg

   Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt, Germany

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Charlott Stock and Lisa Hielkema

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1721-8666

2. Charlott Stock

   Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt, Germany

   Present address

   DANDRITE, Nordic EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Jakob M Silberberg and Lisa Hielkema

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5471-3696

3. Lisa Hielkema

   Department of Structural Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Jakob M Silberberg and Charlott Stock

   Competing interests

   No competing interests declared

4. Robin A Corey

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1820-7993

5. Jan Rheinberger

   Department of Structural Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands

   Contribution

   Formal analysis, Validation, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9901-2065

6. Dorith Wunnicke

   Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt, Germany

   Contribution

   Formal analysis, Validation, Investigation

   Competing interests

   No competing interests declared

7. Victor RA Dubach

   Department of Structural Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1657-7184

8. Phillip J Stansfeld

   School of Life Sciences & Department of Chemistry, University of Warwick, Coventry, United Kingdom

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

9. Inga Hänelt

   Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing

   For correspondence

   haenelt@biochem.uni-frankfurt.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1495-3163

10. Cristina Paulino

    Department of Structural Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands

    Contribution

    Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing

    For correspondence

    c.paulino@rug.nl

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7017-109X

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