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. 2026 Sep 12;23(3):e230030. doi: 10.2142/biophysico.bppb-v23.0030

Structural basis for substrate specificity switching revealed by the FlhA(A489E) suppressor mutation

Motoshi Sakai 1, Miki Kinoshita 2,3, Norihiro Takekawa 1, Keiichi Namba 2,3, Katsumi Imada 1, Tohru Minamino 2,4
PMCID: PMC13639345  PMID: 42840504

Abstract

The flagellar type III secretion system (fT3SS) switches its substrate specificity from hook-type to filament-type upon completion of hook assembly. This process is regulated by the coordinated actions of FliK, FlhB, and FlhA, but the structural mechanism underlying substrate specificity switching remains unclear. Here, we determined the crystal structures of the C-terminal cytoplasmic domain of FlhA(FlhAC) carrying the A489E substitution, which partially restores motility of the cleavage-defective flhB(P270A) mutant. The structures were solved in two distinct conformational states: an open form at 3.28 Å resolution and a semi-closed form at 2.90 Å resolution. In parallel, AlphaFold3-based structural predictions suggested that the flexible C-terminal cytoplasmic tail of FlhB (FlhBCCT) binds to a groove formed between the α2 helix and β2 strand of FlhAC, overlapping with the intersubunit linker-binding site within the FlhAC ring. Structural analyses further revealed that the A489E substitution rearranges a local salt-bridge network involving Arg-386 and Glu-483, leading to subtle positional shifts of the α2 helix and remodeling of the predicted FlhBCCT-binding surface. These structural changes may alter the predicted interaction between FlhAC and FlhBCCT, thereby lowering the energetic barrier associated with conformational remodeling of the FlhAC ring during substrate specificity switching. Together, our results provide structural insight into how local electrostatic remodeling modulates conformational dynamics of FlhAC and regulates substrate specificity switching in the fT3SS.

Keywords: AlphaFold3, flagellar assembly, substrate specificity switching, type III secretion system, X-ray crystallography

Significance

Local electrostatic interactions often control large-scale conformational transitions in biological nanomachines. Here, structural analyses of the FlhA(A489E) suppressor mutant reveal how subtle rearrangements in a salt-bridge network modulate conformational remodeling of the bacterial flagellar type III secretion system (fT3SS). AlphaFold3 predictions further suggest that the flexible C-terminal cytoplasmic tail of FlhB regulates substrate specificity switching by stabilizing the hook-type conformation of the FlhA ring. Our findings provide structural insight into how local interaction networks govern dynamic remodeling of the fT3SS during flagellar assembly.

Introduction

The bacterial flagellum is a supramolecular nanomachine that enables motility and contributes to environmental adaptation and host colonization. Flagellar assembly is driven by the flagellar type III secretion system (fT3SS) that exports structural subunits from the cytoplasm to the distal growing end of the flagellum. During assembly, the fT3SS switches its substrate specificity from hook-type to filament-type upon completion of hook assembly. This substrate specificity switching represents a central checkpoint in flagellar biogenesis and is controlled by the coordinated actions of at least three flagellar proteins, FliK, FlhA, and FlhB [1].

FliK functions as a secreted molecular ruler that monitors hook length and transmits the hook-length signal to the substrate-docking platform formed by the C-terminal cytoplasmic domains of the fT3SS component proteins FlhB (FlhBC, residues 211–383) and FlhA (FlhAC, residues 328–692) to switch the substrate specificity to filament-type once the hook reaches its mature length of approximately 55 nm in Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella) [2]. FlhBC undergoes autocleavage at a conserved NPTH motif (Supplementary Figure S1A) [3,4], and this processing event is essential for switching [5]. FlhAC forms a homo-nonamer [6] and provides binding sites for flagellar export chaperones in complex with their cognate filament-type substrates (Supplementary Figure S1B) [7–10]. Structural and functional analyses have suggested that conformational remodeling of the FlhAC ring is required for the transition from hook-type to filament-type export (Figure 1) [11–14]. However, how the hook-length signal from FliK is transmitted to the substrate-docking platform to trigger the conformational remodeling of the FlhAC ring remains incompletely understood.

Figure 1.

Figure 1

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Model for substrate specificity switching of the fT3SS. During hook assembly, the substrate-docking platform formed by the C-terminal cytoplasmic domains of FlhA (FlhAC) and FlhB (FlhBC) adopts the hook-type state and transports hook-type substrates. When the hook reaches its mature length of approximately 55 nm, the C-terminal export-switch domain of FliK (FliKC) transiently interacts with FlhBC and FlhAC. These interactions promote dissociation of FlhBC from the FlhAC ring and structural remodeling of the FlhAC ring from the hook-type to the filament-type state. The resulting filament-type FlhAC ring provides a functional binding site for filament-type chaperone–substrate complexes, thereby terminating hook assembly and initiating filament assembly at the hook tip. The outer membrane (OM), peptidoglycan layer (PG), and cytoplasmic membrane (CM) are indicated.

Previous studies showed that substitutions at the FlhBC autocleavage site, such as P270A, impair substrate switching and result in polyhook formation [5]. Suppressor analyses of the flhB(P270A) mutant identified a compensatory mutation in FlhAC, in which Ala-489 in the D1 domain is replaced by glutamate (A489E) [15]. Ala-489 is located adjacent to Thr-490, a residue involved in binding filament-type chaperone-substrate complexes (Supplementary Figure S1B) [8], suggesting that the A489E substitution may influence FlhAC ring remodeling. Although the A489E mutation partially restores filament-type export in the presence of defective FlhBC cleavage, the molecular mechanism underlying this suppression effect has remained unclear.

In this study, we determined the crystal structures of FlhAC (A489E) in two distinct conformational states, an open form and a semi-closed form, at resolutions of 3.28 Å and 2.90 Å, respectively. In parallel, AlphaFold3-based structural predictions were used to analyze interactions between FlhAC and the flexible C-terminal cytoplasmic tail of FlhBC (FlhBCCT). By integrating experimental and computational approaches, we reveal how the A489E substitution remodels local electrostatic interactions and modulates conformational dynamics of FlhAC. Our findings provide structural insight into the dynamic regulatory mechanism underlying substrate specificity switching in the fT3SS.

Materials and methods

Structural modeling of the Salmonella FlhAC-FlhBC complex

Structural models of the Salmonella FlhAC-FlhBC complex were generated using the AlphaFold3 prediction server (https://alphafoldserver.com) [16].

Bacterial strains and media

Bacterial strains and plasmids used in this study are listed in Supplementary Table S1. LB medium contained 10 g of Bacto-Tryptone, 5 g of yeast extract and 5 g of NaCl per liter. Soft tryptone agar plates contained 10 g of Bacto Tryptone, 5 g of NaCl and 0.35% (w/v) Bacto-Agar per liter. Ampicillin was added as needed at a final concentration of 100 μg ml–1.

Protein expression and purification

Escherichia coli BL21 (DE3) (Novagen) cells carrying pYI104-SP3, which encodes FlhAC(A489E) with an N-terminal His-tag, were grown overnight at 30°C in 2 L of LB medium supplemented with ampicillin. His-FlhAC(A489E) was purified by affinity chromatography using a HisTrap HP 5 mL column (Cytiva), followed by size-exclusion chromatography on a Superdex 75 10/300 GL column (Cytiva), as described previously [17]. Peak fractions were collected and concentrated to 10 mg mL–1 using an Amicon Ultra-15 centrifugal filter device (Merck Millipore).

Crystallization of His-FlhAC(A489E)

Initial crystallization screening was performed using the sitting-drop vapor-diffusion method with commercially available screening kits [Wizard Classic I–IV and Wizard Cryo I–II (Rigaku, Tokyo, Japan)] at 4°C or 20°C. Drops were prepared by mixing 0.5 μL of protein solution (10 mg mL–1) with 0.5 μL of reservoir solution and equilibrated against 90 μL of reservoir solution. Crystallization conditions were optimized by varying precipitant concentrations, pH, and additives.

Form 1 crystals were grown at 20°C using a reservoir solution containing 0.1 M K2HPO4/Na2HPO4 (pH 6.2), 10% (w/v) polyethylene glycol 3000, and 10% (w/v) glycerol. Form 2 crystals were grown at 20°C using a reservoir solution containing 0.1 M HEPES-NaOH (pH 7.5), 20% (w/v) polyethylene glycol 3000, and 200 mM NaCl. Drops were prepared by mixing 1 μL of protein solution with 1 μL of reservoir solution and equilibrated against 1 mL of reservoir solution.

Data collection and structural determination

X-ray diffraction data were collected at beamlines BL41XU and BL45XU at SPring-8 (Sayo, Japan) with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal Nos. 2019A/B2551 and 2021A2739). Crystals were cryoprotected, flash-frozen in liquid nitrogen, and maintained at –173°C during data collection.

Diffraction data were processed using MOSFLM [18] and scaled with AIMLESS [19]. Initial phases for Form 1 and Form 2 crystals were obtained by molecular replacement using Phaser-MR [20] in Phenix [21] with PDB entries 6AI0 and 3A5I, respectively. Model building was performed using COOT [22], followed by iterative cycles of manual model building and refinement with Phenix [21]. Data collection and refinement statistics are summarized in Supplementary Table S2. Atomic coordinates and structure factors of the Form 1 (semi-closed form) and Form 2 (open form) of FlhAC(A489E) have been deposited in the Protein Data Bank under accession codes 23TK and 23TM, respectively.

Sequence conservation analysis of FlhAC was performed using the ConSurf web server (https://consurf.tau.ac.il) [23].

Structural comparisons and figure preparation were performed by UCSF ChimeraX [24].

Soft-agar motility assays

Fresh colonies were inoculated onto soft agar plates supplemented with ampicillin and incubated at 30°C. Each assay was performed at least seven times to confirm the reproducibility of the results.

Secretion assays

Salmonella cells were grown in 5 ml LB medium with supplemented with ampicillin at 30°C with shaking until the cell density reached an OD600 of approximately 1.2–1.4. After centrifugation, the whole cellular and culture supernatant fractions were prepared as described previously [25]. Protein samples were separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting using polyclonal anti-FlgD or anti-FliC antibody. Immunoblotting was performed with an iBind Flex Western Device according to the manufacturer’s instructions (Thermo Fisher Scientific). Chemiluminescent signals were detected using Amersham ECL Prime Western Blotting Detection Reagent (Cytiva) and captured with a Luminoimage Analyzer LAS-3000 (GE Healthcare). The assay was performed at least three times to confirm the reproducibility of the results.

Results

AlphaFold3 predicts the interaction between FlhAC and FlhBC

Certain mutations in FlhBC autonomously switch the substrate specificity of the fT3SS from hook-type to filament-type in the absence of FliK [26,27]. Because substrate specificity is determined by the conformational state of the FlhAC ring (Figure 1) [11–14], this observation raises the possibility that FlhBC prevents premature structural remodeling of the FlhAC ring from the hook-type state to the filament-type state.

To examine this possibility, we analyzed the predicted structure of the FlhAC-FlhBC complex using AlphaFold3 (Figure 2B and Supplementary Figures S1C and 2) [16]. Confidence assessment of the highest-ranked model showed high pLDDT values for most structured regions of both FlhAC and FlhBC, whereas lower confidence was observed in flexible regions (Supplementary Figure S3A). The PAE map also indicated uncertainty in the relative positioning of these flexible regions (Supplementary Figure S3B). Notably, the orientation of the N-terminal α-helix of FlhBC varied among the five models, consistent with the structural flexibility of this region observed in previous crystallographic and molecular dynamics analyses (Supplementary Figure 1B) [28]. Despite this conformational variability, all five independently generated models consistently positioned FlhBCCT at a groove formed between the α2 helix and β2 strand of the D1 domain of FlhAC, as well as at the loop connecting helices α1 and α2 (Figure 2B and Supplementary Figures S1C and 2).

Figure 2.

Figure 2

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Interaction between FlhAC and FlhBC. (A) Crystal structure of the open conformation of FlhAC (PDB ID: 3A5I). FlhAC (residues 328–682) is composed of four domains (D1–D4) and a flexible linker region (FlhAL) connecting the N-terminal transmembrane domain and FlhAC. The asymmetric unit contains two FlhAC molecules, Mol-A (rainbow) and Mol-B (light grey). FlhAL of Mol-B (magenta) interacts with helix α2 in domain D1 of Mol-A. (B) AlphaFold3 prediction of the FlhAC-FlhBC complex. The C-terminal cytoplasmic domain of FlhB (FlhBC, residues 211–383) contains a flexible C-terminal cytoplasmic tail (FlhBCCT, residues 352–383), which is predicted to interact with a groove formed between the α2 helix and β2 strand of domain D1 in FlhAC as well as with the loop connecting the α1 and α2 helices.

Notably, the α2–β2 groove overlaps with the binding site for the flexible linker region of FlhAC (FlhAL, residues 328–361; Figure 2A) derived from the neighboring FlhAC subunit in the filament-type ring assembly [11,29]. Previous studies have shown that Glu-351 and Asp-356 within FlhAL interact with a positively charged cluster on the α2 helix and play a critical role in FliK-dependent remodeling of the FlhAC ring [13,14]. The predicted FlhAC-FlhBC binding model therefore suggests that FlhBCCT and FlhAL compete for overlapping interaction surfaces on FlhAC.

Consistent with this model, deletion of FlhBCCT has been reported to permit autonomous switching from hook-type to filament-type protein export in the absence of FliK [30]. Together, these observations support the hypothesis that FlhBCCT stabilizes the hook-type conformation of the FlhAC ring by occupying the α2-β2 groove and thereby suppressing spontaneous conformational remodeling of the FlhAC ring.

Overall structures of FlhAC(A489E) in open and semi-closed conformations

The P270A mutation in FlhBC prevents FlhBC autocleavage within the conserved NPTH motif, resulting in the formation of polyhooks [5]. This phenotype suggests that FlhBC(P270A) cannot efficiently dissociate from FlhAC when the hook reaches its mature length of approximately 55 nm in Salmonella. The A489E substitution in the α4 helix of domain D1 of FlhAC can bypass the FlhBC(P270A) defect, thereby increasing the probability of filament formation at the polyhook tip. Consequently, the motility of the flhB(P270A) mutant carrying the flhA(A489E) mutation is improved compared with that of the flhB(P270A) mutant [15]. The predicted FlhAC-FlhBC complex shows that Pro-270 in FlhBC is spatially distant from Ala-489 in FlhAC (Supplementary Fig. 1C), suggesting that the A489E substitution suppresses the FlhB(P270A) defect through an indirect structural mechanism rather than through a direct interaction between these two residues.

To elucidate the structural basis of the suppressor activity of the A489E mutation, we determined the crystal structures of FlhAC(A489E) in two distinct conformational states (Figure 3 and Supplementary Figure S4). The open conformation (PDB ID: 23TM, right) was solved at 3.28 Å resolution, whereas the semi-closed conformation (PDB ID: 23TK, left) was determined at 2.90 Å resolution (Supplementary Table S2).

Figure 3.

Figure 3

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Effects of the FlhA(A489E) substitution on the intramolecular salt bridge between Arg-386 and Glu-483 in the semi-closed and open conformations of FlhAC revealed in the crystal structures. Arg-386 and Ile-390 are located within the α2 helix of domain D1, whereas Ala-489 is located within the α4 helix of domain D1. In the wild-type structures (blue), this residue, together with Ile-390, stabilizes the orientation of the Arg-386 side chain through hydrophobic contacts, thereby allowing formation of an intramolecular salt bridge with Glu-483 in both the semi-closed (PDB ID: 6AI0, left) and open (PDB ID: 3A5I, right) conformations. In the FlhAC(A489E) structures (cyan), the A489E substitution induces local rearrangement of the side-chain interaction network surrounding residue 489. In the semi-closed conformation (PDB ID: 23TK, left), Arg-386 forms a salt bridge with Glu-489 but not with Glu-483. In contrast, in the open conformation (PDB ID: 23TM, right), Arg-386 simultaneously forms salt bridges with both Glu-489 and Glu-483, generating an alternative electrostatic interaction network that is not observed in the wild-type structure. The structures were aligned using domain D3 as a common structural reference and are shown in the same orientation to facilitate comparison of the local side-chain conformations.

Both structures exhibit the canonical four-domain architecture (D1–D4) characteristic of FlhAC (Supplementary Figure S4). Superposition of the A489E structures with previously reported wild-type FlhAC structures revealed that the overall fold is well conserved, with root-mean-square deviations of 0.777 Å and 0.498 Å for the semi-closed and open conformations, respectively.

To understand how the A489E substitution promotes conformational switching of the FlhAC ring from the hook-type state to the filament-type state in the presence of the flhB(P270A) mutation, we analyzed local side-chain interaction networks surrounding residue 489 (Figure 3 and Supplementary Figure S5).

In the wild-type semi-closed structure, Ala-489 is positioned adjacent to Arg-386 and, together with Ile-390, stabilizes the side-chain orientation of Arg-386 through hydrophobic contacts. As a result, Arg-386 adopts a conformation that favors formation of a salt bridge with Glu-483 in domain D2, thereby stabilizing the semi-closed conformation. Interestingly, these four residues are evolutionarily conserved among 150 bacterial species (Supplementary Figure S6), suggesting an important role for this interaction network in FlhA function.

In contrast, in the FlhAC(A489E) semi-closed structure, Arg-386 is displaced from Glu-483 (4.76–9.83 Å) while remaining close to Glu-489 (2.48–3.73 Å) in the two molecules in the asymmetric unit (Supplementary Figure S5). Thus, the introduced Glu-489 establishes a new electrostatic interaction with Arg-386, altering the orientation of the Arg-386 side chain and disrupting its original interaction with Glu-483.

In the wild-type open structure, Ala-489, together with Ile-390, also contributes to stabilization of the Arg-386 side chain through hydrophobic contacts. Arg-386 consequently forms a salt bridge with Glu-483 in domain D2, thereby creating a functional binding site at the interface between domains D1 and D2 for flagellar export chaperones complexed with their cognate filament-type substrates.

In the open conformation of FlhAC(A489E), Arg-386 remains close to both Glu-483 and Glu-489, with interatomic distances of 2.85–2.86 Å and 2.80–3.65 Å, respectively, in the two molecules in the asymmetric unit (Supplementary Figure S5). These interactions generate an alternative electrostatic network that is not observed in the wild-type structure and may allow FlhAC(A489E) to preserve the functional chaperone-binding site even in the presence of the flhB(P270A) mutation.

These observations indicate that the A489E substitution remodels the local electrostatic interaction network surrounding Arg-386, thereby altering the relative stability of the semi-closed and open conformations. Such subtle rearrangements may facilitate both conformational remodeling of the FlhAC ring from the hook-type state to the filament-type state and preservation of the functional chaperone-binding site at the D1–D2 interface under conditions where FlhB dissociation is impaired. Together, these findings suggest that the A489E substitution may lower the energetic barrier between these conformational states, thereby shifting the conformational equilibrium of the FlhAC ring toward the filament-type state.

Rearrangement of the FlhAC-FlhBC interaction surface induced by the A489E substitution

FlhAC undergoes dynamic domain motions through a conserved GYXLI motif (residues 368–372) in domain D1 [31]. This conserved motif is directly involved in substrate specificity switching of the fT3SS from the hook-type to filament-type [14]. In the semi-closed conformation, the orientation of domain D4 relative to domain D3 is intermediate between those observed in the previously reported open and closed conformations [12]. This intermediate state is therefore considered to represent a structural transition state during FlhAC ring remodeling.

We next investigated whether the local electrostatic interaction network around Arg-386 induced by the A489E substitution also alters the FlhAC-FlhBC interaction surface in the semi-closed form of FlhAC(A489E) (Figure 4 and Supplementary Figure S7). Because intersubunit D3-D3 interactions form the core of the FlhAC ring structure [32], we superimposed domain D3 (residues 504–583) of the semi-closed structure of FlhAC(A489E) with that of the wild-type. The RMSD between 80 pruned atom pairs was 0.485 Å. Localized conformational differences were observed in the D1 domain, particularly in the vicinity of the α2 and α4 helices (Supplementary Figure S7A). As a result, several side chains on the α1 and α2 helices exhibit altered rotamer conformations (Supplementary Figure S7B).

Figure 4.

Figure 4

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Effect of the FlhA(A489E) substitution on local side-chain interaction networks in domain D1 of the semi-closed FlhAC structure. (A) Structural comparison of the GYXLI motif in wild-type FlhAC (PDB ID: 6AI0, left, blue) and FlhAC(A489E) (PDB ID: 23TK, right, cyan). Domain D3 of FlhAC(A489E) was superimposed onto that of wild-type FlhAC to visualize relative positional changes. Gly-368, Tyr-369, Arg-370, Leu-371, and Ile-372 constitute the GYXLI motif, which is highlighted in magenta in the ribbon representation, with its side chains shown as stick models colored by element. This GYXLI motif is responsible for cyclic open-to-closed domain motions [31]. Genetic evidence suggests that the C-terminal export-switch domain of FliK may bind to this motif to induce conformational remodeling of the FlhAC ring from the hook-type state to the filament-type state [14,33]. (B) Structural comparison of local side-chain interaction networks within the groove formed between the α1 and α2 helices and β2 strand of wild-type FlhAC (left) and FlhAC(A489E) (right). AlphaFold3 predicts that the flexible C-terminal cytoplasmic tail of FlhBC interacts with this groove (Figure 2).

Genetic evidence suggests that FliK not only interacts with the GYXLI motif in domain D1 to induce structural remodeling of the FlhAC ring [14,33] but also promotes dissociation of FlhBC from the FlhAC ring [34]. Interestingly, the A489E substitution induces local remodeling of side-chain interaction networks surrounding the GYXLI motif (Figure 4A). Furthermore, the A489E substitution weakens local hydrophobic interaction networks within the groove formed between the α1 helix and β2 strand (Figure 4B). Because this groove is predicted to constitute a major component of the predicted FlhBCCT-binding surface (Figure 2), these structural changes presumably alter the interaction between FlhAC and FlhBCCT.

Because FliK remains required for conformational remodeling of the FlhAC(A489E) ring [15], we propose that formation of the intramolecular Arg-386–Glu-489 salt bridge facilitates FliK-dependent remodeling of the FlhAC ring in the presence of the flhB(P270A) mutation. This remodeling may stabilize an alternative electrostatic interaction network involving Arg-386, Glu-483, and Glu-489, thereby favoring formation of the filament-type FlhAC ring containing the functional chaperone-binding site at the D1–D2 interface.

Role of the conserved Arg-386 residue for flagellar protein export

Arg-386 is highly conserved among 150 bacterial species (Supplementary Figure S6). To clarify its biological role in flagellar protein export, we replaced this residue with alanine and performed complementation assays using a Salmonella ΔflhA strain as the host. FliH and FliI are required for efficient activation of the export gate complex through the interaction of FliJ with FlhA [35]. Furthermore, FliH and FliI contribute to ensuring the correct order of flagellar protein export during flagellar assembly [14,36]. In fact, FlhA(A489E) requires FliH and FliI for efficient protein export [15]. Because the flhB(P28T) bypass mutation increases the probability of flagellar formation in the absence of FliH and FliI, we also used a Salmonella ΔfliH-fliI flhB(P28T) ΔflhA strain to examine the role of Arg-386 under conditions in which ATPase-dependent export gate activation is impaired. Flagella-driven motility and fT3SS-mediated protein secretion were assessed by soft-agar motility assays and immunoblotting using polyclonal antibodies against FlgD as a representative hook-type export substrate and FliC as a representative filament-type export substrate (Figure 5).

Figure 5.

Figure 5

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Role of the highly conserved Arg-386 residue in flagellar protein export. (A) Soft-agar motility of Salmonella [ΔflhA, indicated as ΔA] (left) NH001, NH004 [ΔfliH-fliI flhB(P28T) ΔflhA, indicated as ΔHI-B* ΔA] (middle), or NH002 [flhB(P28T) ΔflhA, indicated as B* ΔA] (right) carrying pTrc99AFF4 (V, vector control; a modified pTrc99A expression vector), pMM130 (WT), or pMKM130(R386A). Plates were incubated at 30°C. Data are representative of at least seven independent experiments. (B) Secretion of FlgD and FliC. Whole-cell (Cell) and culture supernatant (Sup) fractions were prepared from the transformants shown in (A) and analyzed by immunoblotting with the indicated polyclonal antibodies. Molecular mass markers (kDa) are indicated. Data are representative of at least three independent experiments.

Expression of FlhA(R386A) fully restored motility (Figure 5A, left panel) and flagellar protein export (Figure 5B, left panel) in the presence of FliH and FliI. In contrast, the flhA(R386A) substitution inhibited both motility (Figure 5A, middle panel) and protein export (Figure 5B, middle panel) in the absence of FliH and FliI. The flhB(P28T) mutation alone had no apparent effect on either motility (Figure 5A, right panel) or protein export (Figure 5B, right panel). These results indicate that Ala-386 retains the ability to form a functional interaction network with Ile-390, Glu-483, and Ala-489 in the presence of FliH and FliI but not in their absence.

These results suggest that Arg-386 becomes particularly important for efficient flagellar protein export when FliH/FliI-dependent activation of the export gate is impaired. Because the cytoplasmic ATPase complex promotes dynamic structural remodeling of the FlhAC ring [14,37], the electrostatic interaction network involving Arg-386 may contribute to maintaining conformational dynamics of the FlhAC ring for efficient and robust protein export during flagellar assembly.

Discussion

In this study, we determined the crystal structures of FlhAC(A489E) in the open and semi-closed conformations and investigated how the A489E suppressor mutation influences substrate specificity switching of the fT3SS. Our structural analyses, combined with AlphaFold3-based modeling and functional assays, suggest that local remodeling of an electrostatic interaction network surrounding Arg-386 modulates conformational dynamics of the FlhAC ring and facilitates formation of the filament-type export state.

Previous studies demonstrated that conformational remodeling of the FlhAC ring is required for substrate specificity switching from hook-type to filament-type [11–14]. In particular, the intersubunit FlhAL binding region of FlhAC plays an essential role in stabilizing the filament-type ring conformation through interactions with a positively charged surface surrounding the α2 helix [11,13,33]. In the present study, AlphaFold3 predictions suggest that FlhBCCT occupies a partially overlapping binding surface on FlhAC (Figure 2). This model provides a plausible explanation as to how FlhBC stabilizes the hook-type state of the FlhAC ring prior to hook completion.

The flhB(P270A) mutation, which interferes with autocleavage of FlhBC at the conserved NPTH motif, prevents substrate specificity switching of the fT3SS and results in the formation of polyhooks with or without filaments [5]. This phenotype suggests that autocleavage of FlhBC is required for efficient dissociation of FlhBC from FlhAC upon hook completion. The flhA(A489E) mutation was previously isolated as an extragenic suppressor of the flhB(P270A) mutant [15]. In the present study, we found that the A489E substitution induces subtle but reproducible rearrangements in local side-chain interaction networks involving Arg-386, Ile-390, and Glu-483 (Figure 3). These structural changes alter the orientation of the α2 helix and surrounding residues that contribute to the predicted FlhBCCT-binding surface (Figure 4). Because the semi-closed conformation likely represents a transition-state intermediate during FlhAC ring remodeling [12], the altered electrostatic interaction network observed in FlhAC(A489E) may lower the energetic barrier associated with conformational switching of the FlhAC ring.

Our findings also suggest a functional relationship between ATPase-dependent export gate activation and conformational dynamics of the FlhAC ring. The R386A substitution had only minor effects on flagellar protein export in the presence of FliH and FliI but strongly impaired export in their absence (Figure 5). These observations suggest that the FliH-FliI complex promotes dynamic conformational remodeling of the FlhAC ring required for efficient flagellar protein export, thereby reducing the requirement for precise electrostatic stabilization within the ring. Thus, the local interaction network surrounding Arg-386 may become particularly important when FliH/FliI-dependent remodeling of the FlhAC ring is compromised.

Based on these findings, we propose a model in which FlhBC stabilizes the hook-type state of the FlhAC ring by occupying the α2–β2 groove prior to hook completion (Figure 6A). In this model, the hook-type state is defined as the FlhAC ring associated with FlhBC, in which FlhAL has not yet engaged the neighboring FlhAC subunit. When the hook reaches its mature length of approximately 55 nm, FliKC interacts with FlhBC and the conserved GYXLI motif in domain D1 of FlhA, triggering conformational remodeling of the FlhAC ring. This remodeling promotes dissociation of FlhBC from FlhAC and allows FlhAL to interact with the neighboring FlhAC subunit, thereby stabilizing the filament-type ring structure with a functional chaperone-binding site at the D1–D2 interface.

Figure 6.

Figure 6

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Structural remodeling of the FlhAC ring during substrate specificity switching of the fT3SS. (A) Model for structural remodeling of the FlhAC ring. During hook assembly, FlhBC (plum) binds to domain D1 of FlhAC, thereby stabilizing the hook-type state of the FlhAC ring. When the hook reaches its mature length of approximately 55 nm, FliKC interact with the GYXLI motif in domain D1 and with FlhBC, allowing FlhAC to adopt a transient intermediate state. FliKC then promotes dissociation of FlhBC from FlhAC, allowing FlhAL to interact with the neighboring FlhAC subunit. As a result, the FlhAC ring adopts the filament-type conformation, thereby terminating hook assembly and initiating filament assembly at the hook tip. (B) Proposed rescue model for the FlhB(P270A) defect by the FlhA(A489E) substitution. In the wild type, interactions of FliKC with FlhBC and FlhAC are proposed to efficiently lower the energetic barrier for the transition from the hook-type to the filament-type state. The FlhB(P270A) substitution impairs this FliK-dependent lowering of the energetic barrier, thereby inhibiting the transition to the filament-type state. In the FlhB(P270A)/FlhA(A489E) background, the FlhA(A489E) substitution partially restores FliK-dependent lowering of the barrier, thereby increasing the probability of transition to the filament-type state, although the transition remains less efficient than in the wild type. The energy landscapes shown are schematic representations of the proposed energetic barriers and do not represent experimentally determined free-energy profiles.

We further propose that the FlhB(P270A) substitution impairs this FliK-dependent remodeling process by maintaining a relatively high energetic barrier between the hook-type and filament-type states (Figure 6B). The A489E substitution remodels the local electrostatic interaction network surrounding Arg-386 and partially facilitates FliK-dependent lowering of this barrier. Consequently, FlhA(A489E) can undergo the transition toward the filament-type state in the presence of FlhB(P270A) more efficiently than wild-type FlhA, although switching remains less efficient than in the wild-type FlhB background. Thus, the A489E substitution appears to suppress the FlhB(P270A) defect by modulating the conformational energy landscape of the FlhAC ring rather than through a direct interaction with Pro-270.

Collectively, our results provide structural insight into how local electrostatic interactions regulate large-scale conformational transitions in the fT3SS. These findings further highlight the importance of dynamic interaction networks in controlling assembly-coupled substrate specificity switching in bacterial nanomachines.

Conclusion

In summary, our structural and functional analyses reveal that the FlhA(A489E) suppressor mutation remodels a local electrostatic interaction network surrounding Arg-386 and alters conformational dynamics of the FlhAC ring. Together with AlphaFold3-based predictions of the FlhAC-FlhBC interaction, our findings support a model in which dynamic remodeling of local interaction networks regulates substrate specificity switching in the fT3SS. These results provide a new insight into how local structural perturbations control global conformational transitions in a biological nanomachine.

Conflict of interest

The authors declare that there is no conflict of interests.

Author contributions

K.N., K.I., and T.M. conceived and designed research; M.K. and T.M. performed genetic, biochemical, and physiological experiments; M.S., N.T., and K.I. prepared protein samples and performed crystallization, crystallographic data collection, processing, refinement, and model building. K.N., K.I., and T.M. wrote the paper based on discussion with other authors.

Data availability

The evidence data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Coordinates, structure factors and its validation report of the Form 1 (semi-closed form) and Form 2 (open form) of FlhAC(A489E) were deposited to the Protein Data Bank under the PDBID: 23TK with the DOI of https://doi.org/10.2210/pdb23TK/pdb. and the PDBID: 23TM with the DOI of https://doi.org/10.2210/pdb23TM/pdb.

Acknowledgements

We thank Yasuyo Abe for technical assistance and beamline staff at SPring-8 for technical help in use of beamlines BL41XU and BL45XU. This work was supported in part by JSPS KAKENHI Grant Numbers JP20K15749 and JP22K06162 (to M.K.), JP16J01859 and JP23K14158 (to N.T.), JP15H02386 and 21H02443 (to K.I.), JP19H03182, JP22H02573, and JP22K19274 (to T.M.). This work has also been supported by Research Support Project for Life Science and Drug Discovery (BINDS) from AMED under Grant Number JP23am121003, JP24am121003 and JP25am121003 (to K.N.), by the Cyclic Innovation for Clinical Empowerment (CiCLE) from AMED under Grant Number JP17pc0101020 (to K.N.), and by JEOL YOKOGUSHI Research Alliance Laboratories of The University of Osaka (to K.N.).

References

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Materials (4.9MB, pdf)

Data Availability Statement

The evidence data generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Coordinates, structure factors and its validation report of the Form 1 (semi-closed form) and Form 2 (open form) of FlhAC(A489E) were deposited to the Protein Data Bank under the PDBID: 23TK with the DOI of https://doi.org/10.2210/pdb23TK/pdb. and the PDBID: 23TM with the DOI of https://doi.org/10.2210/pdb23TM/pdb.

Articles from Biophysics and Physicobiology are provided here courtesy of The Biophysical Society of Japan

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