Molecular mechanism of phosphate import by the bacterial PstSCAB transporter

Domain: neuroscience
Published: 2026-02-03
Editorial outcome: minor_revision

Abstract
Inorganic phosphate (Pi) is essential for all living organisms. PstSCAB, a bacterial high-affinity ABC transporter, imports Pi under limiting conditions via five subunits: PstA and PstC forming the transmembrane domain (TMD), periplasmic PstS that switches between free and TMD-docked forms for Pi capture and delivery, and two cytosolic PstB subunits for ATP binding and hydrolysis. Its malfunction affects the virulence of pathogenic bacteria, making it pharmaceutically attractive. However, complete structural pictures of PstSCAB in different states remain lacking. Here, we determine cryo-EM structures of PstSCAB in resting, pretranslocation, and catalytic intermediate states, which reveal that conformational changes in PstS and ATP binding/unbinding in PstB collectively induce rigid-body movements of TMD, generating inward- or outward-facing conformations. In TMD, Pi specificity is determined by positively charged Arg220 (PstA) and Arg237 (PstC). This study advances understanding of bacterial Pi import and supports drug development targeting PstSCAB.

Introduction
Phosphorus, the fifth most abundant element in cells, is essential for all living organisms. At the cellular level, phosphorus plays a role in the inheritance of genetic materials, energy metabolism, membrane integrity, and intracellular signaling. At the macroscopic level, misregulation of phosphorus can result in human disorders1, decreased agricultural productivity2, and lethality in microorganisms3, etc., therefore, it is of medical, agricultural, and pharmaceutical significance.

Inorganic phosphate (Pi) is the main form of phosphorus available in the environment, and different organisms have evolved distinct Pi translocating systems for the exchange of Pi across cell membranes. In Escherichia coli (E. coli), two Pi transporter systems are involved in Pi import: the low-affinity inorganic phosphate transporter (PiT) system, and the high-affinity phosphate-specific transport (Pst) system4. Under an excessive Pi environment, Pi is responsible for Pi uptake, and Pst is repressed, whereas under Pi-limiting conditions, Pst is activated. Conditional activation of PstSCAB is possible because it is part of the Pho regulon controlled by a two-component regulatory system comprised of a membrane-spanning histidine kinase sensor protein (PhoR) and a cytosolic response regulator (PhoB)5. Under Pi limitation, PhoR functions as a kinase that phosphorylates PhoB, allowing PhoB to bind to promoters with a consensus DNA sequence called the PHO box and regulate the expression of genes in the Pho regulon. When Pi is abundant, PhoR functions as a phosphatase that dephosphorylates PhoB, causing the dissociation of PhoB from downstream promoters and the reversal of gene expression regulation. Although the exact signal sensed by PhoR is not clear, extracellular Pi concentration has been ruled out, as PhoR does not seem to contain a periplasmic sensory domain; instead, alternative conformations in PstSCAB have been proposed as a possible signal6, probably involving PhoU that is encoded in the same operon (PstSCAB-phoU operon)7.

In addition to the genes involved in maintaining phosphate homeostasis, the Pho regulon contains numerous virulence genes associated with quorum sensing, chemotaxis, toxin secretion, and regulation, which contribute to changes in virulence-related phenotypes, such as biofilm formation, intestinal colonization, motility, and cytotoxicity. Therefore, pathological or experimental disruption of PstSCAB function has been shown to compromise its control on the Pho regulon and thereby the virulence of pathogenic bacteria8–12. For example, in urinary tract infections (UTI) causing Proteus mirabilis, PstS and PstC homologs were identified as virulence factor required for colonization of the urinary tract11. Some studies showed that mutations of PstA/C affected host cell adherence by atypical enteropathogenic E. coli (EPEC) and virulence of EPEC-like murine pathogen Citrobacter rodentium8. In another study adopting a functional genomics approach, PstS and PstC were among the identified virulence factors involved in Edwardsiella tarda pathogenesis12. Inactivation of the Pst system attenuated the virulence of Avian pathogenic E. coli (APEC) strain chi7122 in a chicken infection model10. In addition, PstSCAB is directly or indirectly involved in the pathogenicity of the septicemia-inducing E. coli strain in calves and piglets, and disruption of PstC by transposon abolished its pathogenicity9. More recently, it was shown that PstS from Mycobacterium tuberculosis (Mtb) PstSCAB is targeted by antibodies isolated from Mtb-infected patients13, implying its role in Mtb pathogenesis.

In PstSCAB transporter, PstS, a phosphate-binding protein with a two-lobed structure, is the periplasmic subunit that captures Pi from the periplasm and then delivers them to the substrate binding site in the transmembrane domain (TMD) formed by PstA and PstC. Therefore, PstS has to undergo conformational changes between the open, Pi-free state and closed, Pi-bound state. Two cytosolic PstB subunits form the nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, providing the energy needed to release TMD-bound Pi into the cytosol. Although the periplasmic subunit PstS has been subjected to extensive structural studies13–21, the architecture of the whole PstSCAB complex and its structures in different functional states remain elusive, leaving the molecular mechanism of Pi specificity and Pi import in TMD unaddressed.

Here, we determine the cryo-electron microscopy (cryo-EM) structures of PstSCAB in resting, inward-facing/pretranslocation, and outward-facing/catalytic intermediate states. Along with ATPase assay, MST assay, Pi import assay, and molecular dynamics (MD) simulations, our study sheds light on the molecular mechanism of Pi specificity and import in PstSCAB.

Results
Biochemical characterization and structure determination
In E. Coli, all 4 subunits in PstSCAB are encoded in an operon named PstSCAB-PhoU. We initially tried to purify E. coli PstSCAB transporter from BL21 (DE3) cells overexpressing the whole PstSCAB operon, utilizing an affinity tag on PstB, which only yielded a complex without PstS (referred to as PstCAB hereafter) (Fig. 1a). We then changed strategy and reconstituted PstSCAB complex using PstCAB and PstS that were expressed and purified separately, with an E179Q mutation in PstB that prevents ATP hydrolysis. Using ATPase assays, we showed that while the wild-type PstCAB displayed robust ATPase activity, the ATPase activity for PstCAB E179Q was largely abolished (Fig. 1b, c). Substrate inhibition effect was observed at higher ATP concentrations (Fig. 1c), which was also observed in “Bce-type” ABC transporter BceAB from Bacillus subtilis22. Such inhibition is likely due to accumulation of Pi from ATP hydrolysis, as higher concentrations of Pi also showed inhibitory effect (Fig. 1d). Surprisingly, addition of PstS did not have notable effects on ATPase activity of PstCAB (Fig. 1e), unlike the stimulatory effect of substrate-binding proteins (SBPs) observed in other bacterial ABC importers such as maltose transporter23, and E. coli cysteine/cystine ABC transporter YecSC-FliY24. Additionally, using MST assays, we demonstrated that PstCAB could bind Pi, with a Kd of 7.76 mM (Fig. 1f).

By incubating PstCAB and PstS with ATP, we managed to obtain the PstSCAB complex in an ATP-bound, catalytic intermediate state (referred to as PstSCAB E179Q_ATP hereafter) (Supplementary Fig. 1a). However, we did not see binding between PstCAB and PstS in the absence of ATP. To capture PstSCAB in pretranslocation state (ATP unbound form), we inspected the interfaces between PstS and PstA/C in its ATP-bound outward-facing structure (described in detail later) and introduced two pairs of cysteine residues in N-lobe and C-lobe of PstS, and periplasmic loops of PstA and PstC, respectively (PstS N48C/K167C and PstC D63C/E149C, referred to as “4Cys” hereafter), followed by cross-linking with Dithio-bismaleimidoethane, with wild-type PstB subunits and in the absence of ATP, which yielded PstSCAB 4cys representing pretranslocation state (Supplementary Fig. 1b).

Purified PstCAB, PstSCAB 4Cys and PstSCAB E179Q_ATP were reconstituted into nanodiscs and then subjected to single-particle cryo-EM analysis. 3D reconstructions yielded 4 high-resolution maps in 3 distinct conformations, namely resting, pre-translocation, and catalytic intermediate states, at overall resolutions of 3.0/3.3, 2.9, and 2.7 Å respectively (Supplementary Figs. 2–4 and Table 1). For the PstSCAB dataset, two 3D reconstructions corresponding to inward-facing PstCAB and outward-facing PstSCAB E179Q_ATP conformations were obtained during 3D classification. Overall, the quality of the electron densities is superb for most part of the complex, allowing accurate model building (Supplementary Fig. 5). The only exception is PstB, which can be reliably modeled only in PstSCAB E179Q_ATP, but not in PstCAB and PstSCAB 4Cys, owing to its poor density. Therefore, PstB in PstCAB and PstSCAB 4Cys was built by rigid-body fitting of its coordinates from PstSCAB E179Q_ATP. The ATP-free, inward-facing PstCAB maps obtained from PstCAB and PstSCAB 4Cys are indistinguishable from each other (Supplementary Fig. 6a). Therefore, the one from PstCAB with higher resolution was deposited and used for discussion.

Structure of the resting state PstCAB
In the resting state, the transporter presumably has just gone through conformational changes that opened its Pi-binding site in the membrane-spanning subunits to the cytosol and released the bound Pi into the cell, and is ready for another Pi import cycle. In agreement with our biochemical data, the inward-facing structure consists of one copy of PstA and PstC, and two copies of PstB, but not PstS (Fig. 2a, b). This is similar to the resting state structure of MBP-MalFGK2 and Methanosarcina acetivorans ModBC25, whose SBPs are not docked, but different from those of trehalose importer LpqY-SugABC26, oligopeptide permease OppABCD27 and heme/tri- or tetra-peptides importer DppABCD28 from Mycobacterium tuberculosis (Mtb), whose SBPs are already associated with TMD in the resting state (Supplementary Fig. 7).

Both PstA and PstC contain six transmembrane helices (TMs) (Fig. 2c, d). Similar to ModB2C2 and MBP-MalFGK2, two subunits in the membrane (PstA and PstC) are intertwined, with TM1 of one subunit contacts with TM2-TM6 of the other subunit (Fig. 2e). Although of limited sequence homology (~19% sequence identity), the structure of PstA and PstC are similar to each other, with an root mean square deviation (RMSD) of 1.78 Å over 231 residues when superimposed, and are therefore related to each other via a 2-fold pseudo symmetry (Fig. 2e). PstA and PstC form a transmembrane heterodimer in the membrane, with a cavity that faces the cytosol and is inaccessible from the periplasm, owing to a closed gate formed by several patches of conserved residues (“gating regions”) (Fig. 2f). Residues in these “gating regions” are located at the periplasmic tip of TM5 from both PstA (Ala226, Leu229, and Phe230) and PstC (Thr242, Met243, and Phe247), and the loop between TM5 and TM6 in PstA (Phe254) and PstC (Ala272), right above the Pi binding site in the membrane (see later sections) (Fig. 2f). This gating regions ensure that the Pi binding site, which is located in the interface between PstA and PstC, is accessible only from the cytosol.

Two PstBs bind to PstA and PstC, one for each, through a coupling apparatus conserved among type I ABC transporters (e.g., a groove formed in the ATP-binding subunits and a coupling helix within the EAA loops of membrane-spanning subunits) (Fig. 2g, h). Like the counterparts in other bacterial ABC transporters, PstB forms a NBD, which can be further divided into RecA-like subdomain containing Walker A and B motifs and a helical subdomain containing the LSGGQ signature motif (Fig. 2b), essential for ATP binding. For some ABC transporters, e.g., MBP-MalFGK and Methanosarcina acetivorans ModBC, there is a regulatory domain in addition to NBD, which forms contact with that of the neighboring subunit (Supplementary Fig. 7). In PstSCAB, since no such regulatory domain is present, its resting state structure is held together solely by interactions between PstA and PstC (with a buried surface area of ~3468 Å2), which explains that the poor density at the distal end of PstBs is due to their flexibility (Fig. 2a).

Structure of PstSCAB in pretranslocation state
To capture the structure of PstSCAB in the pretranslocation state, we had to introduce cysteines into the interface between PstS and PstA/PstC, followed by crosslinking, implying that the pretranslocation (pre-T in short hereafter) state is probably transient (ATP binding and hydrolysis likely happen shortly after Pi-loaded PstS docks onto PstA/C). TMD of the pre-T state structure is inward-facing conformation, similar to that of the resting state structure (Fig. 3a, b). One copy of closed, Pi-bound PstS docks on TMD from the periplasmic side (Fig. 3a, b).

As has been shown in previous structural characterization, PstS is structurally similar to other cluster D-IIIa SBPs and adopts a two-lobed structural fold that is shared among the periplasmic components of bacterial ABC type importers29 (Fig. 3b and Supplementary Fig. 7). Structure of PstS in the pre-T state is similar to the isolated crystal structure of PstS in Pi-bound closed conformation (pdb#1ixh), with an RMSDs of 0.69 Å over 319 residues when superimposed (Supplementary Fig. 8a). In PstS structure of pre-T state, an extra non-protein density corresponding to Pi is observed in the cavity between the two lobes, which overlaps with bound Pi in the previously reported PstS crystal structure in Pi-bound form (Fig. 3c and Supplementary Fig. 8a). This observation consolidates our claim that this structure is in the pre-T state, in which PstS is loaded with Pi and is ready to release its bound Pi into the Pi binding site in the membrane (in the outward-facing or catalytic intermediate state, see below).

PstS docks onto PstA and PstC in a manner that its N-lobe sits above PstA, whereas its C-lobe sits above PstC (with a buried surface area of ~749 and ~606 Å2 respectively) (Fig. 3b). Between PstA and the N-lobe of PstS, Asp 21, Lys 30 and Asn 32 in PstS form hydrogen bonds with Asn 235, Gln 236 and Trp 238 in PstA respectively (Fig. 3d). Between PstC and the C-lobe of PstS, Lys 175 in PstS forms hydrogen bond with Asn 273 in PstC (Fig. 3f). In addition, Val 31, and Thr 49 in PstS form interactions with Cys 149, and Phe 237 in PstA (Fig. 3d). While the interactions between PstS and PstA/C seem sparse and occurred only at one side of each interface, two long loops between TM1 and TM2 (P1 loop), and TM5 and TM6 (P3 loop) in both PstA and PstC, extend upward into the cleft between N-lobe and C-lobe of PstS, and form additional interactions with them, which further stabilize PstSCAB complex (Fig. 3d–f). In PstA, while Pro 73 in P1 loop form hydrophobic interactions with the C-lobe of PstS (e.g., Gln 201), Pro 260 and Phe 261 in P3 loop form hydrophobic interaction with the C-lobe of PstS (e.g., Gln 201 and Asn 202) (Fig. 3e). Similarly, in PstC, Asn 273, Glu 277 and Ser 280 in P3 loop form interactions with the N-lobe of PstS (e.g., Lys 43 and Lys 175) (Fig. 3f).

Surprisingly, the structures of PstA, PstC and PstB are almost identical between the resting and pre-T state, with an RMSD of 1.25 Å over 923 residues (Fig. 3a, b and Supplementary Fig. 6b). This is similar to LpqY-SugABC, but different from MBP-MalFGK2, as MalF and MalG were brought closer in the pre-T state upon binding of maltose-bound MBP, which is an intermediate between resting and outward-facing states (Supplementary Fig. 7)30. Other transient pre-T intermediate states may exist, where PstS adopts intermediate conformations between closed and open states. Transition to catalytic intermediate states is probably initiated by conformational changes in PstS, since two PstBs are too far away to jointly bind ATPs and initiate such transition. Potentially, in these pre-T intermediate states not captured in this study, two PstB monomers will be close enough to allow joint ATP binding by two PstBs, further accelerating the conformational transition of PstSCAB into an outward-facing conformation.

Structure of PstSCAB in ATP-bound, outward-facing state
In the ATP-bound state of PstSCAB, the binding of ATPs tightly glues two copies of PstB together, resulting in the outward-facing conformation of the whole transporter complex that allows the release of Pi from PstS into the Pi binding site in TMD (Fig. 4a, b). PstB binds ATP in a manner that is conserved in ABC transporters, involving residues in the Walker A and B motifs from one monomer and the LSGGQ motif of the opposite monomer (Fig. 4c). The docked PstS is in open, ligand-free conformation, highly similar to the previously reported crystal structure of PstS in the ligand-free state (PDB#1oib), with a RMSD of 1.13 Å over 317 residues when superimposed (Supplementary Fig. 8b). The interfaces between PstS and TMD are dramatically different from that of pre-T state, involving completely different set of residues (Fig. 4d, e). For example, interactions are formed between Pro 71, Tyr 141, Ser 234 in PstA and Asp 21, Asn 32, Gln 34, Lys 43, Thr 44 in the N-lobe of PstS (Fig. 4d). On the other hand, Leu 132, Asn 251, Gln 254, Asn 273, in PstC are involved in forming interactions with residues in the C-lobe of PstS (Arg 134, Gly 138, Pro 169, Lys 175 and Arg 186) (Fig. 4e). In addition, Met 258 & Pro 260 in PstA, and Ala 276 and Glu 277 in PstC form interactions with residues in C-lobe (Tyr 198 and Gln 201) and N-lobe (Ser 38 and Ser 39) of PstS (Fig. 4d, e), further sealing the cavity between PstS and TMD. Consistent with the stable binding of PstS to TMD in the catalytic intermediate state, the buried surface areas are ~964 and ~909 Å2 between N-lobe PstS and PstA, and C-lobe PstS and PstC, respectively, as compared to ~749 and ~606 Å2 in the pre-T state. The increase in buried surface area seems to be mainly contributed by PstS (Supplementary Fig. 9). Moreover, dramatic conformational changes occurred in transmembrane subunits (PstA and PstC) and their interacting ATP-binding modules (PstBs), as will be discussed in detail later.

A density is observed in the middle of TMD in the cryo-EM map of the outward-facing conformation of PstSCAB, inside the helical bundle formed by TM4 and TM5 of both PstA and PstC, but not in the inward-facing state and pre-T state, likely representing Pi (Fig. 4f). Although the density does not fit perfectly with a Pi molecule, several pieces of evidences support the identity of this density as being Pi. First, location of this density in PstSCAB is similar to the bound maltose in MBP-MalFGK2, as well as trehalose in LpqY-SugABC, all positioned in a cavity in the interface between two TMD subunits (Supplementary Fig. 7). Second, residues that are in coordination distance with this density are mostly positively charged, with their side chains pointing toward the above mentioned density, which agrees well with the favored chemical environment for Pi binding (see below for details), similar to the Pi binding environment in XPR131. Moreover, as reported previously, mutations of some of these residues were shown to abolish the Pi transport activity, whereas the ATPase activity remained unaffected32–34. Third, the modeled Pi binds stably in the pocket throughout a 1 μs MD simulation (Fig. 4g and Supplementary Fig. 10). To rigorously assess Pi-binding stability, we analyzed the distances between Pi and its coordinating residues. Pi forms stable contacts with PstA Ser130, PstA Arg220, and PstC Arg237. The time-dependent distance profiles further confirm this claim: the distances from Pi to PstA Arg220 and PstC Arg237 are exceptionally stable, showing minimal fluctuation over the entire simulation (Supplementary Fig. 11). The distance to PstA Ser133 exhibits more variation but remains within a bound state. In addition, we further calculated the root-mean-square fluctuation (RMSF) for both Cα atoms and side chains (averaged per residue) of the Pi-neighboring residues (Supplementary Fig. 12). The results confirm that the Cα-RMSF for all neighboring residues is very low, while the side chain RMSF shows a certain mobility.

Pi is coordinated by residues from both PstA and PstC, e.g., the side chain of Arg 220 and the main chain of TM5 from PstA, as well as the side chain of Arg 237 and the main chain of TM5 from PstC (Fig. 4f). Earlier studies have shown that mutation of PstA Arg220 and PstC Arg237 largely compromised the Pi transport activity32–34. Moreover, mutation of PstC Glu241 also abolished Pi transport activity. According to our structure, PstC Glu241 forms salt bridge with PstC Arg237, thereby stabilizing the Pi binding site (Fig. 4f). Pseudo-symmetrically, PstA Glu224 also forms salt bridge with PstA Arg220 (Fig. 3f), although its mutation did not have any effects on Pi transport activity32. Using MST assays, we show that mutations of its coordinating residues largely reduced the Pi binding affinity of PstCAB (Fig. 4h and Supplementary Table 2). Moreover, in our Pi import assays, mutation of these residues by Crispr-cas9 editing in E. coli resulted in largely impaired Pi import activity when compared with that of the wild-type PstSCAB (Fig. 4i).

Structural transitions among different states
Dramatic structural changes were observed among PstSCAB structures in different states. While the main structural difference between PstCAB and PstSCAB is the absence or presence of PstS in PstCAB, structural changes occur broadly within the complex when comparing PstSCAB 4cys and PstSCAB E179Q_ATP (Fig. 5a).

Specifically, PstS switches from a compact conformation in PstSCAB 4cys (the cleft between two lobes of PstS is closed, with Pi engulfed inside it) to a loose conformation in PstSCAB E179Q_ATP (the cleft between two lobes of PstS is open, without bound Pi), around the inter-domain hinge (Fig. 5b). When the N-lobes of PstS from these two states were aligned, the C-lobe of PstS in PstSCAB E179Q_ATP swings ~15° away from the central cleft (Fig. 5b). Two PstBs undergo large conformational changes, involving both translational and rotational movements (Fig. 5c, d). As a result, two PstBs are brought closer to each other in PstSCAB E179Q_ATP, allowing joint binding of two ATPs in the resulting dimer interface (Fig. 5c, d). In TMD, the gate at periplasmic side is closed and the gate at cytoplasmic side is open in the structure of PstSCAB 4cys, therefore it represents the inward-facing, pre-T state (Fig. 5e). By contrast, in TMD of PstSCAB E179Q_ATP, periplasmic half of PstA and PstC, which are directly coupled with two lobes of PstS, move away from the center of two-fold pseudo-symmetric axis, whereas their cytoplasmic half move towards the center of two-fold pseudo-symmetric axis (Fig. 5f). Consequently, the gate at periplasmic side becomes dilated and the gate at cytoplasmic side is constricted, thereby exposing the binding site in the membrane to the periplasm instead of the cytosol, which is the basis of alternative access model of most transporters (Fig. 5e, f and Supplementary Fig. 13). Cytoplasmic gate is mainly formed by TM4 and TM5 of PstA and PstC, which can move conveniently along with PstB, as the connecting helix between TM4 and TM5 (the coupling helix) in both PstA and PstC is responsible for forming direct interaction with PstB (Figs. 2g, h and 5e, f). Similar to other ABC transporters, conformational changes in TMD are achieved by rigid-body movements in PstA and PstC, particularly their TM2-TM5 (Fig. 5g, h).

Methods
Cloning and overexpression of PstS and PstSCAB
All primers used in this study have been listed in Supplementary Table 3. The protein-coding sequence of pstS and pstSCAB, which are contained in the pstSCAB-phoU operon, were amplified from E. coli DH5α genomic DNA by PCR, and cloned into pET28a(+), using either conventional digestion and ligation (pstS, digested with NcoI/XhoI) or Gibson assembly (pstSCAB). These constructs allowed the expression of target proteins fused with a 6×His tag at the C terminus. For preparing the inward-facing, pre-translocation state and outward-facing, catalytic intermediate state samples, constructs for expressing PstS N48C/K167C and PstC D63C/E149C A B or PstSCAB E179Q were prepared following the standard protocol of site-directed mutagenesis, and verified by sequencing (Sangon Biotech, China).

The resulting plasmids were transformed into E. coli BL21 (DE3). Single colonies were cultured in Luria broth (LB) with 50 μg/mL kanamycin for 12 h at 37 °C. The resulting starter culture was transferred into 1 L LB medium containing kanamycin (50 μg/ml), and incubated at 37 °C until the OD reached 0.8. Then, 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added, and the induction continued at 16 °C for 20 h. Cells were harvested by centrifugation, flash-frozen in liquid nitrogen, and stored at −80 °C.

Protein purification
To purify the PstCAB/PstCAB E179Q complex, frozen cell pellets were re-suspended in lysis buffer containing 50 mM Tris, pH 8.0, 200 mM NaCl and 1 mM PMSF (buffer A), and lysed by using a high-pressure homogenizer (JNBIO, Guangzhou) at 1000 bar for 4 cycles (same procedure for cell lysis throughout the method section). Unbroken cells and large debris were removed by centrifugation at 10,000 × g for 30 min at 4 °C, and 1% (w/v) Lauryl maltose neopentyl glycol (LMNG, Anatrace) were added into the supernatant for membrane protein extraction (gentle agitation for 2 h at 4 °C). After centrifuging at 4 °C, 20,000 × g for 30 min, the membrane protein-containing supernatant was applied to a disposable column with 1 mL Nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (GE Healthcare). After extensive wash steps, the protein was eluted with 20 mM Tris, pH 8.0, 150 mM NaCl (buffer B) containing 0.01% (w/v) LMNG and 200 mM imidazole, concentrated, and purified using a Superose 6 Increase column (GE Healthcare) in buffer B containing 0.006% (w/v) LMNG. Protein fractions with the highest homogeneity were collected and concentrated to ~4 mg ml−1 and stored at −80 °C.

To obtain the PstSCAB complex, PstS N48C/K167C and PstC D63C/E149C A B protein were purified separately. The cell pellets of PstS N48C/K167C were re-suspended in phosphate-buffered saline (PBS) (pH 7.2) containing 1 mM PMSF and lysed. Unbroken cells and large debris were removed by centrifugation at 10,000 × g for 30 min at 4 °C. The supernatant was loaded on Ni-NTA agarose beads (GE Healthcare) affinity column and incubated for 30 min at 4 °C followed by elution with PBS buffer containing 200 mM imidazole. The protein was then loaded into a Superdex 75 Increase column equilibrated with PBS, and peak fractions were pooled. PstC D63C/E149C A B was purified following a similar protocol to PstCAB/PstCAB E179Q except for using PBS buffer containing 1% (w/v) LMNG for membrane protein extraction. PstS N48C/K167C and PstC D63C/E149C A B were then incubated in PBS buffer containing 0.01% LMNG (w/v) at a molar ratio of 1:1 with the presence of 1 mM dithio-bismaleimidoethane (DTME, Thermo Scientific) for 1 h at room temperature. Successful crosslinking was confirmed by treatment with 100 mM dithiothreitol (DTT, Thermo Scientific) followed by SDS-PAGE electrophoresis (Supplementary Fig. 1b). The crosslinked products were then purified using Superose 6 Increase column equilibrated with PBS, pH 7.2 containing 0.006% (w/v) LMNG.

To obtain the PstSCAB complex, PstS was purified following a similar protocol to PstS N48C/K167C, except for using lysis buffer containing buffer A. The purification procedure for the PstCAB E179Q complex was described above. The protein samples (PstS:PstCAB E179Q at a molar ratio of 2:1) were incubated with 1 mM ATP, 1 mM MgCl2, and 2 mM HPO4 2− for 30 min at room temperature, followed by size-exclusion chromatography on a Superose 6 Increase column (GE Healthcare) in buffer B containing 0.006% (w/v) LMNG to yield a stable complex.

For Nanodisc reconstitution, PstCAB, PstSCAB 4Cys or PstSCAB E179Q_ATP, MSP1D1 membrane scaffold protein, and POPG were mixed at a molar ratio of 1:1:30 in buffer B containing 0.006% (w/v) LMNG and incubated for 1 h at 4 °C. Detergents were removed by incubation with 50 mg ml−1 Bio-Beads SM2 (Bio-Rad) for 1 h at 4 °C, followed by centrifugation. For the resulting supernatant, the same procedure was repeated 3 more times (incubation period of 1 h, 1 h, and overnight, respectively). Nanodisc-embedded PstCAB, PstSCAB 4Cys or PstSCAB E179Q_ATP were further purified using a Superose 6 Increase column in buffer B.

Cryo-EM sample preparation and data acquisition
For the PstCAB, PstSCAB E179Q_ATP and PstSCAB 4cys samples, Quantifoil R1.2/1.3 (300 mesh) holey carbon grids were first glow discharged for 50 s using a Pelco easiGlow glow discharge unit, and 4 μL sample was applied to the surface of the grid at a temperature of 5 °C and humidity level of 100%. Grids were then blotted for 2.0 s before being plunge-frozen in liquid ethane using Vitrobot Mark IV. The cryo-specimens were loaded onto a Titan Krios transmission electron microscope (Thermo Fisher) operated at 300 kV for data collection. The microscope is equipped with a GIF-Quantum energy filter (Gatan), which was used with a slit width of 20 eV. Automatic data collection was performed using EPU software. Images were recorded with Gatan K3 direct electron detectors operating in super-resolution counting mode at pixel sizes of 0.856, 0.856 and 0.67 Å, respectively. The exposure was performed with a dose rate of 15 e-/pixel/s and an accumulative dose of ~50 e-/Å2 for each image, fractionated into 36 movie-frames. The final defocus ranges of the datasets were approximately −(1.2–2.5) μm.

Image processing and 3D reconstruction
For the PstCAB data, the dose-fractionated image stacks were subjected to beam-induced motion correction using MotionCor249. Initial contrast transfer function (CTF) values for each micrograph were calculated with CTFFIND450. Micrographs with an estimated resolution limit worse than 6 Å were discarded in the initial screening. About 10,000 particles were blob-picked and subjected to 2D classification to generate templates for auto-picking against the entire dataset. The subsequent image processing and reconstruction were performed using cryoSPARC51. 3,228,106 particles were picked from 6777 micrographs. Then the picked particles were extracted and subjected to three rounds of reference-free 2D classification in cryoSPARC, which yielded 1,194,741 particle projections. This subset was subjected to one round of ab-initio reconstruction followed by Heterogeneous Refinement. The predominant class containing one subset of 579,652 best particles shows the clear features of secondary structural elements. These particles were subjected to a non-uniform refinement, which yielded a reconstruction at 3.0 Å resolution (Supplementary Fig. 2).

The PstSCAB E179Q_ATP and PstSCAB 4cys datasets were processed similarly to the above PstCAB data. The detailed image processing and reconstruction are shown in Supplementary Figs. 3 and 4. Local resolution estimate was performed with cryoSPARC.

Model building
Model building was manually conducted in Coot52. Accurate residue registration was achieved based mainly on the clearly defined densities for bulky residues (Phe, Trp, Tyr, and Arg). Models were refined against cryo-EM maps using real-space refinement in PHENIX53, with secondary structure and non-crystallography symmetry restraints applied. The statistics of the models’ geometries were generated using MolProbity54. Ion translocating pathways were calculated using Caver 3.055. All the figures were prepared using PyMol56 or ChimeraX57.

ATPase assay
All ATPase assays were performed using the ATPase/GTPase Activity Assay Kit (Sigma-Aldrich). Purified PstCAB and PstCAB E179Q (1 μg) were incubated in a 20-μl reaction volume in buffer B containing 1 mM ATP and 1 mM MgCl2 (Sangon Biotech) for 10 min at 37 °C, which represents the standard reaction system. For measurement of concentration-dependent ATPase activity, varying concentrations of ATP were used. For the effect of Pi and PstS, varying concentrations of Pi and PstS were added in the standard reaction system. After the reaction was stopped and the colorimetric product was generated, absorbance at 620 nm was measured at room temperature using a SpectraMax iD3 multifunction reader (Molecular Devices). ATPase activity was represented as phosphate (nanomoles) produced by 1 mg of protein per minute.

Microscale thermophoresis (MST) assays
The PstCAB complex and its relevant mutants were prepared following a similar protocol to PstCAB/PstCAB E179Q except for using the Hepes buffer (pH 7.4). Protein labeling was performed using the Monolith His-Tag Labeling Kit RED-tris-NTA 2nd Generation (Nanotemper Technologies, Cat# MO-L018). Specifically, 90 μL of His-tagged proteins (200 nM) and 90 μL of dye (100 nM) were mixed and incubated for 30 min at room temperature under no-light conditions. Following centrifugation at 15,000 × g for 10 min at 4 °C, the labeled protein was immediately subjected to phosphate-binding assays using the Monolith NT.115 (NanoTemper Technologies). The assay mixture containing labeled proteins and varying concentrations of Pi was loaded into the Monolith NT.115 capillaries (Nanotemper Technologies, Cat# MO-K022). The MST measurements were performed with 40% LED/excitation power and high MST power. All experiments were performed in triplicate. Data analysis was carried out using the manufacturer’s analysis software (NanoTemper software).

Gene editing
A modified CRISPR/Cas9 system (pEcCas/pEcgRNA) was used for gene knock-out and knock-in in E. coli strain TOP10, following the procedure described previously58. In brief, the pEcCas plasmid, encoding Cas9 and λ-Red recombinase, was used to facilitate homologous recombination, with λ-Red expression induced by 10 mM L-arabinose. The pEcgRNA plasmid, carrying the sgRNA and an N20-specific sequence, was employed to target the gene for editing. Target-specific 20 bp spacers were predicted using the CHOPCHOP website (https://chopchop.cbu.uib.no). Homologous arms, 500 bp upstream and downstream of the target gene, were linked via overlapping extension PCR to create the knockout donor template. The strain carrying pEcCas was cultured at 37 °C until the OD 600 reached 0.2, after which 10 mM arabinose was added to induce λ-Red recombinase expression. The culture was kept at 37 °C until the OD 600 reached 0.8, after which competent cells were prepared. These cells were electroporated with 200 ng of pTarget plasmid and 800 ng of donor DNA, and transformants were plated on LB agar containing 50 μg/mL kanamycin and 50 μg/mL spectinomycin after 1 h of incubation at 37 °C. Positive transformants were confirmed by colony PCR and DNA sequencing. The pTarget plasmid was cured using 10 mM rhamnose, and the pEcCas plasmid was eliminated by adding 10 mM sucrose.

Phosphate uptake measurements
Procedure for Pi uptake measurement is adapted from a previous study6. Two types of medium were used: MOPS (morpholinepropanesulfonic acid), MOPS with 0.4% glucose and 0.1 mM Pi (referred to as MOPS LoPi). Cells were grown overnight in 5 ml MOPS LoPi. After washing twice with 5 ml MOPS medium, the cells were resuspended to an OD 600 of ~0.45 in MOPS medium containing 0.4% glucose, but no Pi. The Pi-starved cells were incubated at 37 °C on a roller drum for 2 h. For transport assays, 750 μl of cells at an OD 600 of 0.45 were added to 750 μl of a 10.5 μM solution of KH2PO4. After incubating for 45 s at room temperature, 1000 μl of cells were removed and filtered through pre-wet 0.2 μm nitrocellulose filters. Using the Malachite Green assay (Sigma), Pi concentrations were measured for each filtrate sample. The net amount of Pi (in nmoles) in the filtrate (the amount in a blank reaction containing no cells was subtracted) was divided by the dry weight of cells in each sample and the time, which yielded the Pi uptake rate. Each strain was assayed using three biological replicates in triplicate.

Molecular dynamics simulations
Systems for the PstSCAB structure in the catalytic intermediate state were built using the CHARMM-GUI server59, with each structure embedded in a palmitoyl-oleyl-phosphatidylcholine bilayer and hydrated with 50,662 TIP3P water molecules60. The final systems were neutralized with the sodium ion, and extra NaCl was added to model the experimental salt concentration of 0.15 M, resulting in a simulation box approximately 110 Å × 110 Å × 173 Å in dimension, encompassing a total of 216,586 atoms. All simulations were carried out using GROMACS-2025.2 software package61 with the CHARMM36m force field62. The van der Waals cutoff was set to 12 Å and PME was used for long-range electrostatic energy calculations. These systems were first minimized for 50,000 steps with the steepest descent algorithm, followed by an equilibration process at 300 K for 1125 ps in NPT ensemble. The final production simulations were performed at 300 K under the NPT ensemble with the integration step of 2 fs. The stochastic velocity rescaling method63 and the Parrinello–Rahman method64 were used for temperature and anisotropic pressure coupling, respectively. Three independent runs with the identical starting structures and randomized initial velocities were carried out to improve samplings, with each trajectory lasting for 1 μs. All data were analyzed using the facilities from GROMACS-2025.2 software package and all figures were prepared using gnuplot 5.4. The RMSD values were computed relative to the initial structure with Cα atoms. Supplementary Fig. 10 shows that the systems converged rapidly. Accordingly, data from the 400–1000 ns time interval were selected for all subsequent analyses to ensure converged calculations.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed all of my previous concerns. The revised manuscript now presents a clearer and more transparent cryo-EM analysis, along with improved model refinement statistics. In addition, all minor issues have been appropriately addressed. However, the terminology referring to the “electron density map” remains incorrect and must be revised. The authors should replace “electron density map” with “cryo-EM density map” in the legends of Figures 2, 3, and 4 before the manuscript can be accepted for publication.", "reviewer_2": "The authors have addressed my comments and clearly made an effort to expand the analysis of the catalytic state. However, much of this expanded analysis appears primarily in the response letter. It is unclear whether these additions will be incorporated into the revised manuscript or remain solely in the response.\nRegarding the results presented in the main text and supplementary materials, I would like to offer a suggestion for a clearer presentation and interpretation.\nIn Supplementary Fig.10, the RMSD of the overall structure from the initial conformation appears to be around 0.2 nm (2 Å), which indicates relatively stable behavior. In contrast, Supplementary Fig. 11 shows certain residue–residue distances fluctuating by approximately 3–6 Å or more from the initial structure. These residues are located in what I understand to be a highly constrained region, and such a large deviation seems inconsistent with the overall RMSD of ~2 Å. Could the authors please verify this measurement? Additionally, for the stability of Pi and the surrounding residues. I recommend calculating mean square fluctuations (MSF) after equilibration to quantify the fluctuations of key residues identified as functionally important with respect to the others. This would more accurately reflect structural/dynamic stability.\nFinally, this work is primarily experimentally driven, and I will therefore largely defer to other reviewers on the experimental aspects. While the structural snapshots effectively highlight key functional events, I would recommend including a concise summary of the computational results, even if limited to the catalytic state. A brief discussion to contextualize these findings, together with potential computational extensions, would help provide a more complete perspective.", "reviewer_3": "The authors have satisfactorily addressed my concerns and comments. The provided movie more clearly illustrates the entire phosphate (Pi) transport process. The ATPase activity data of PstSCAB4Cys, which is comparable to that of PstCAB, have partially alleviated my concerns regarding whether the PstSCAB4Cys structure genuinely represents the authentic pre-translocation state in vivo. However, Point 10 from my previous critique still requires further attention and revision to ensure full scientific clarity. Repeated experiments indicate that the PstCE241QAR220QB mutant continues to exhibit high variability. Could this be attributed to abnormal protein expression? Given the unreliability of these results, should a reasonable explanation be included in the article, or should the data be removed altogether to avoid misleading readers?"}, "author_response": "Authors’ Reply: We thank the reviewer for positive evaluation. We have replaced\n“electron density map” with “cryo-EM density map” in the legends of Figures 2, 3, and\n4.\n\nAuthors’ Reply: We thank the reviewer for the positive comments on our efforts. Here,\nwe have chosen to present the newly added analyses of the catalytic state in the response\nletter, as a preliminary investigation into the allosteric effects in this system, rather than\nincorporating them into the main text. This decision is based on the following rationale:\n(1) While our current analyses have yielded valuable insights into the system's intrinsic\ndynamics by probing the catalytic state, we fully agree with the reviewer's original\ncomment that a more ideal approach would involve investigating all three functional\nstates—the pre-translocation state, the catalytic intermediate, and the resting state. This\nwould allow for a more comprehensive understanding of the system's dynamic\nregulation. Including only the dynamics of the catalytic state in our experimentally\nfocused manuscript would render the presentation incomplete, less definitive, and\npotentially bias the focus of the current study. (2) As acknowledged by the reviewer,\nthis study is primarily experimentally driven. It is difficult to incorporate these\nsimulation data into this manuscript without affecting its integrity. Therefore, we\nbelieve it is more scientifically sound to conduct a complete and dedicated\ncomputational study that treats all three resolved states with equal emphasis, aiming to\nelucidate the transition pathways and deliver more meaningful and profound dynamic\ninsights. This comprehensive analysis is, in fact, the focus of our ongoing work.\nRegarding the results presented in the main text and supplementary materials, I would\nlike to offer a suggestion for a clearer presentation and interpretation.\nIn Supplementary Fig.10, the RMSD of the overall structure from the initial\nconformation appears to be around 0.2 nm (2 Å), which indicates relatively stable\nbehavior. In contrast, Supplementary Fig. 11 shows certain residue–residue distances\nfluctuating by approximately 3–6 Å or more from the initial structure. These residues\nare located in what I understand to be a highly constrained region, and such a large\ndeviation seems inconsistent with the overall RMSD of ~2 Å. Could the authors please\nverify this measurement?\nAuthors’ Reply: We appreciate the reviewer's careful observation. First, we would like\nto clarify that the overall RMSD reported in Supplementary Fig. 10 was calculated\nusing Cα atoms, which reflects the stability of the protein backbone. In contrast, the\nresidue-Pi distances in Supplementary Fig. 11 were measured as the minimum distance\nbetween any atom pairs of two residue groups, including side chains. This metric can\nexhibit significant fluctuations due to side-chain reorientations or torsional adjustments,\neven when the backbone remains stable. We have updated the caption of Supplementary\nFig. 11 to clarify the presented data.\nSecond, we isolated all instances where distance fluctuations exceeded 4.0 Å. The\nresidues and their fluctuation ranges are: S130 (2.4–7.5 Å), A131 (2.6–8.6 Å), and P132\n(3.3–8.2 Å) in Run1; and A131 (2.4–6.4 Å) and P132 (2.6–8.6 Å) in Run2. All other\nfluctuations remained below 4.0 Å. We have now performed a statistical analysis of the\nmean and standard deviation (std) for all Pi-neighboring residue distances across the\nsimulations, as shown in the figure below. Although the absolute maximum fluctuation\nin some frames can reach ~6.0 Å, the primary deviation from the mean, as quantified\nby the std, generally falls within a much narrower range.\nThird, taking Run1 as an example, we extracted two structural snapshots corresponding\nto the minimum (2.6 Å) and maximum (8.6 Å) distances respectively between Pi and\nA131 as shown below. Visual inspection reveals that Pi is primarily coordinated by\nresidues R220 and R237, located on two helical segments, and the distances from these\nkey residues to Pi remain nearly constant, consistent with the data shown in the above\nhistogram figure. Concurrently, it is evident that the side chains of the arginine residues\nundergo a significant conformational adjustment, thereby displacing the Pi molecule\naway from A131. Thus, while the overall Cα-RMSD indicates a stable protein scaffold\nand Pi remains coordinated by a stable set of residues (primarily via side chains), side-\nchain motions can indeed lead to significant distance variations between Pi and other\nneighboring residues such as A131.\nThis interpretation is further supported by the RMSF analysis we conducted following\nthe reviewer's suggestion (see below). We calculated both Cα-RMSF and sidechain-\nRMSF (averaged per residue) for all neighboring residues. The results clearly show that\nwhile Cα-RMSF values are very low, indicating a stable backbone, sidechain-RMSF\nvalues are considerably higher, reflecting greater mobility in the side chains.\nTherefore, we conclude that the stable overall Cα-RMSD values in Supplementary Fig.\n10 are not inconsistent with the distance fluctuations observed in Supplementary Fig.\n11. The coordination of Pi is maintained by a stable core of residues (notably R220,\nR237 via their side chains), while side-chain dynamics account for the observed larger\nfluctuations with other neighboring residues.\nAdditionally, we sincerely thank the reviewer for the meticulous critique. Upon re-\nexamination, we identified that the curves originally described as \"average distance\"\nwere in fact calculated using the minimum distance. We have replaced the original\nhistogram with updated plots showing average distances with error bars (Fig. 4g). We\nwould like to emphasize that this correction does not alter any of the original\nconclusions of the manuscript regarding the stability of the Pi coordination environment.\nAdditionally, for the stability of Pi and the surrounding residues. I recommend\ncalculating mean square fluctuations (MSF) after equilibration to quantify the\nfluctuations of key residues identified as functionally important with respect to the\nothers. This would more accurately reflect structural/dynamic stability.\nAuthors’ Reply: Following the reviewer's recommendation, we have calculated the\nroot-mean-square fluctuation (RMSF) for both Cα atoms and side chains (averaged per\nresidue) of the Pi-neighboring residues after equilibration. The results confirm that the\nCα-RMSF for all neighboring residues is very low, while the sidechain RMSF shows\ncertain mobility. Overall, these data support the conclusion that the Pi coordination\nenvironment remains stable, with fluctuations primarily arising from side-chain\ndynamics. We have incorporated this figure into Supplementary materials\n(Supplementary Fig. 12) and updated the main text with a corresponding statement (see\nline 289-293).\nFinally, this work is primarily experimentally driven, and I will therefore largely defer\nto other reviewers on the experimental aspects. While the structural snapshots\neffectively highlight key functional events, I would recommend including a concise\nsummary of the computational results, even if limited to the catalytic state. A brief\ndiscussion to contextualize these findings, together with potential computational\nextensions, would help provide a more complete perspective.\nAuthors’ Reply: We understand the reviewer's perspective that including the dynamics\nof the catalytic state could provide additional details. However, for the reasons stated\nearlier, we believe that presenting results from only the catalytic state is preliminary\nand incomplete. While it would add some information, it lacks the context provided by\nthe other two states and cannot offer a sufficient or consistent description of the\ndynamic transitions and potential allosteric mechanisms operating between all three\nfunctional states. Therefore, we prefer to reserve the full dynamical analysis for a\nsubsequent, more complete and self-contained study. Such work will consistently reveal\nthe dynamics of the PstSCAB system across all states and provide a more rational and\ncomprehensive understanding. Since we decided not to include the data for dynamic\nstudy of the catalytic state in our current manuscript, we have only added a brief outlook\nin the main text discussing potential future computational studies to provide a more\ncomplete perspective, following the reviewer's suggestion. See line 363-365:\n“Ultimately, integrating these structural insights with multi-scale simulations across all\nfunctional states will be crucial for a complete dynamical understanding of the transport\ncycle.”\nAuthors’ Reply: We thank the reviewer for positive evaluation. To avoid misleading\nreaders, we have removed the MST data for the PstC A B mutant from Fig. 4\nE241Q R220Q\nand Table S2."}, {"round": 2, "reviewer_comments": {"reviewer_2": "I do not have furthe comments."}, "author_response": ""}]

Decision letter