Conserved GYXLI Motif of FlhA Is Involved in Dynamic Domain Motions of FlhA Required for Flagellar Protein Export.

Flagellar structural subunits are transported via the flagellar type III secretion system (fT3SS) and assemble at the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhAC consists of domains D1, D2, D3, and D4 and adopts open and closed conformations. Gly-368 of Salmonella FlhA is located within the highly conserved GYXLI motif and is critical for the dynamic domain motions of FlhAC. However, it remains unclear how it works. Here, we report that periodic conformational changes of the GYXLI motif induce a remodeling of hydrophobic side chain interaction networks in FlhAC and promote the cyclic open-close domain motions of FlhAC. The temperature-sensitive flhA(G368C) mutation stabilized a completely closed conformation at 42°C through strong hydrophobic interactions between Gln-498 of domain D1 and Pro-667 of domain D4 and between Phe-459 of domain D2 and Pro-646 of domain D4, thereby inhibiting flagellar protein export by the fT3SS. Its intragenic suppressor mutations reorganized the hydrophobic interaction networks in the closed FlhAC structure, restoring the protein export activity of the fT3SS to a significant degree. Furthermore, the conformational flexibility of the GYXLI motif was critical for flagellar protein export. We propose that the conserved GYXLI motif acts as a structural switch to induce the dynamic domain motions of FlhAC required for efficient and rapid protein export by the fT3SS. IMPORTANCE Many motile bacteria employ the flagellar type III secretion system (fT3SS) to construct flagella beyond the cytoplasmic membrane. The C-terminal cytoplasmic domain of FlhA (FlhAC), a transmembrane subunit of the fT3SS, provides binding sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhAC undergoes cyclic open-close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhAC. However, it remains unknown how it works. Here, we carried out mutational analysis of FlhAC combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhAC by Gly-368 is important for remodeling hydrophobic side chain interaction networks in FlhAC to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly.

INTRODUCTION

Many motile bacteria utilize flagella to swim in viscous liquids and move around on solid surfaces to migrate toward more favorable environments for their survival. The flagellum of Salmonella enterica serovar Typhimurium (here referred to as Salmonella) is divided into three structural parts: the basal body, which acts as a bi-directional rotary motor, the filament, which functions as a helical propeller to produce thrust, and the hook, which is a universal joint connecting the basal body and filament and transmits torque produced by the motor to the filament. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. To construct the flagellum on the cell surface, the flagellar type III secretion system (fT3SS) transports flagellar structural subunits from the cytoplasm to the distal end of the growing flagellar structure (1).

The fT3SS is located at the base of the flagellum and is composed of a transmembrane export gate complex with a stoichiometry of 9 FlhA, 1 FlhB, 5 FliP, 4 FliQ, and 1 FliR and a cytoplasmic ATPase ring complex with a stoichiometry of 12 FliH, 6 FliI, and 1 FliJ (see Fig. S1A in the supplemental material). In addition, FlgN, FliS, and FliT act as export chaperones that escort their cognate substrates from the cytoplasm to the fT3SS (2, 3). The export gate complex utilizes the transmembrane electrochemical gradient of protons (H+) as the energy source to unfold and translocate export substrates across the cytoplasmic membrane (4, 5). The export gate complex requires ATP hydrolysis by the cytoplasmic ATPase ring complex to become an active H+/protein antiporter that couples H+ flow with protein translocation (6–8). The fT3SS also has an Na+-powered backup engine to maintain the flagellar assembly process when the cytoplasmic ATPase ring complex does not function properly (9, 10).

Salmonella FlhA is composed of an N-terminal transmembrane domain with eight transmembrane helices (FlhATM, residues 1 to 327), a compactly folded cytoplasmic domain (FlhAC, residues 362 to 692), and a flexible linker (FlhAL, residues 328 to 361) connecting these two domains (11, 12) (Fig. S1B). FlhATM acts as a dual-ion channel that conducts both H+ and Na+. An interaction between FlhAL and FliJ activates the ion channel of FlhATM, allowing the export gate complex to couple either H+ or Na+ flow with the translocation of export substrate across the cytoplasmic membrane (13). FlhAC forms a homo-nonamer in the fT3SS (14). The FlhAC ring serves as a docking platform for export substrates and flagellar chaperones along with the C-terminal cytoplasmic domain of FlhB and plays an important role in hierarchical protein targeting and export for efficient flagellar assembly (15–21).

FlhAC consists of domains D1, D2, D3, and D4 (Fig. S1B) and adopts open and closed conformations (12, 22). A highly conserved hydrophobic dimple is located at the interface between domains D1 and D2 and is directly involved in substrate recognition (16, 17). Because a large cleft exits in the interface between domains D2 and D4 in the open form of FlhAC, but not in the closed form (Fig. S1C), the chaperone-substrate complexes can bind to the conserved hydrophobic dimple in the open form but not in the closed form (23, 24). For filament assembly, the C-terminal region of FlhAL binds to domains D1 and D2 of its neighboring FlhAC subunit in the FlhAC ring structure to stabilize the open conformation (Fig. S1C, right panel), allowing the chaperone-substrate complexes to efficiently bind to the FlhAC ring (25). During hook assembly, however, FlhAL binds to the conserved hydrophobic dimple of the open form not only to suppress premature docking of the chaperone-substrate complexes to FlhAC but also to facilitate the export of the hook protein FlgE (26). These observations suggest that the open form of FlhAC reflects an active state of the fT3SS. However, little is known about the role of the closed form of FlhAC in flagellar protein export.

The flhA(G368C) mutation inhibits the protein transport activity of the fT3SS at a restrictive temperature of 42°C but not at a permissive temperature of 30°C (27–29). The temperature shift-up from 30°C to 42°C arrests the export of flagellar proteins by the fT3SS with the flhA(G368C) mutation. Molecular dynamics (MD) simulation has shown that the flhA(G368C) mutation restricts cyclic open-close domain motions of FlhAC at 42°C and stabilizes a completely closed conformation of FlhAC (24). Gly-368 is located within the highly conserved GYXLI motif (Fig. 1A). This leads to a plausible hypothesis that the conserved GYXLI motif may be critical for such domain motions of FlhAC coupled with flagellar protein export.

FIG 1

Role of a highly conserved Gly-368 residue of FlhA in dynamic domain motions of FlhAC. (A) Multiple sequence alignments of the conserved GYXLI motif of FlhA homologs. Multiple sequence alignment was carried out using Clustal Omega. Conserved residues are highlighted in red. A highly conserved glycine residue is indicated by an asterisk. UniProt accession numbers: Salmonella, P40729; Vibrio, A0A6F8WT44; Helicobacter, O06758, Caulobacter, Q03845; Bacillus, P35620. (B) Cα ribbon diagrams of the closed (left panel) and open (right panel) forms of FlhAC-G368C obtained by MD simulation. Phe-459 and Gln-498 make hydrophobic contacts with Pro-646 and Pro-667, respectively, in the closed from but not in the open form. The Cα backbone is color-coded from blue to red, going through the rainbow colors from the N to the C terminus.

To clarify this hypothesis, we carried out mutational analysis of FlhAC combined with MD simulation. We provide evidence that the flhA(G368C) mutation stabilizes hydrophobic side chain interactions between domains D1 and D3 and those between domains D2 and D4, thereby suppressing dynamic open-close domain motions of FlhAC, and that its intragenic suppressor mutations induce the remodeling of the hydrophobic interaction networks in FlhAC, allowing FlhAC with the G368C mutation (FlhAC-G368C) to restore the dynamic open-close domain motions.

RESULTS

Effect of the flhA(G368C) mutation on hydrophobic side chain interaction networks in FlhAC.

To address why the flhA(G368C) mutation stabilizes a completely closed form of FlhAC at 42°C, we first compared the interfaces between domains D1 and D3 and those between domains D2 and D4 in the open and closed forms of FlhAC-G368C obtained in the previous MD simulation (24) (Fig. 1B). Met-398 and Gln-498 of domain D1 make hydrophobic contacts with Pro-550 of domain D3 and Pro-667 of domain D4, respectively (left panel). Phe-459 of domain D2 makes a hydrophobic contact with Pro-646 of domain D4 (left panel). Because the hydrophobic interactions between Phe-459 and Pro-646 and those between Gln-498 and Pro-667 are not seen in the open form of FlhAC-G368C (right panel), we propose that the flhA(G368C) mutation may stabilize these two hydrophobic interactions at 42°C, thereby suppressing cyclic open-close motions of FlhAC.

MD simulation of FlhAC with either the G368C/K548C or G368C/F459C/K548C mutation.

The highly conserved Lys-548 residue of FlhA is located at the interface between domains D1 and D3 and contributes to D1-D3 interactions (12) (Fig. 1B). The flhA(K548C) mutation in domain D3 does not affect FlhA function at all. However, the combination of the flhA(G368C) and flhA(K548C) mutations results in a loss-of-function phenotype even at 30°C (24). To clarify why the flhA(G368C/K548C) mutation inhibits flagellar protein export at 30°C, we analyzed the structure and dynamics of FlhAC-G368C/K548C by MD simulation at 27°C for 1.5 μs (Fig. 2A). Domains D1 and D2 behaved like a rigid body in all cases. In contrast, domain D4 became closer to domain D2 through conformational changes of two hinges, one between domains D1 and D3 and the other between domains D3 and D4, when FlhAC switched from an open conformation to a closed conformation. Therefore, we calculated the center-of-mass distance (d24) between domains D2 and D4 during MD simulation (Fig. S2 and Table S1).

FIG 2

MD simulation of FlhAC with either G368C/K548C or G368C/F459C/K548C. (A) Center-of-mass distance between domains D2 and D4 (d) during a 1.5-μs MD simulation. (B) Representative structures of wild-type (blue), FlhAC-G368C (magenta), FlhAC-G368C/K548C (green), and FlhAC-G368C/F459C/K548C (cyan) obtained by MD simulation. Domains D1 and D2 are superimposed. (C) Structural comparisons between completely closed forms of FlhAC-G368C (magenta) and FlhAC-G368C/K548C (green) (left panels) and between the open forms of wild-type FlhAC (blue) and FlhAC-G368C/F459C/K548C (cyan). (D) Interfaces between domains D1 and D3 and between domains D2 and D4 of FlhAC-G368C/K548C.

Unlike FlhAC-G368C, which changes its conformation back and forth between the closed and open forms at 27°C (24), FlhAC-G368C/K548C stopped the dynamic domain motions at 27°C after 0.3 μs and switched its conformation to a completely closed form (Fig. 2A). When we compared the fully closed form of FlhAC-G368C/K548C with that of FlhAC-G368C, there was a significant difference in the orientation of domain D4 relative to domain D3 (Fig. 2B and C, left panel, and Table S2). Unlike FlhAC-G368C, in which hydrophobic interactions between Gln-498 and Pro-667 and those between Phe-459 and Pro-646 stabilize the closed conformation of FlhAC-G368C (Fig. 1B, left panel), the hydrophobic interactions of Pro-667 with Phe-459 and Leu-461 locked the FlhAC-G368C/K548C structure into the fully closed conformation at 30°C (Fig. 2D).

It has been reported that the flhA(F459C) mutation restores motility of the flhA(G368C/K548C) mutant to a considerable degree (24); we analyzed the effect of this mutation on the structure and dynamics of FlhAC-G368C/K548C by MD simulation. The F459C substitution restored the dynamic open-close domain motions of FlhAC-G368C/K548C at 27°C (Fig. 2A). FlhAC-G368C/F459C/K548C also adopted a super-open conformation (Fig. 2C, right panel, and Table S1) as seen in the wild-type structure at 42°C (24). Therefore, we conclude that the cyclic open-close domain motion of FlhAC is required for efficient export of flagellar structural subunits by the fT3SS and that the completely closed form of FlhAC reflects an inactive state of the fT3SS.

Role of the highly conserved GYXLI motif in flagellar protein export by the fT3SS.

We found structural differences in domain D1 when the open and closed forms of FlhAC-G368C were compared (Fig. S3). Gly-368 forms the highly conserved GYXLI motif along with Tyr-369, Arg-370, Leu-371, and Ile-372 (Fig. 1A), which forms a short α-helix (Fig. S3). Cys-368 of FlhAC-G368C made much stronger hydrophobic contacts with Arg-370, Leu-413, and Pro-415 of domain D1 in the completely closed form than in its open form (Fig. 3A). As a result, the G368C mutation induced a significant conformational change of this α-helix (Fig. S3).

FIG 3

Structural comparison between closed (right panels) and open conformations (right panels) of FlhAC-G368C obtained by MD simulation. (A) Hydrophobic interactions of Cys-368 with Arg-370, Leu-413, and Pro-415 in the open and closed forms of FlhAC-G368C. (B) Hydrophobic interactions between Met-365 and Leu-492. (C) Interaction between Gln-477 and Ala-446.

The flhA(R370S) mutation, which has been isolated as an intragenic suppressor mutation of the flhA(G368C) mutant grown at 42°C (28), is located within the conserved GYXLI motif (Fig. 1A). When the open and closed forms of FlhAC-G368C obtained by MD simulation were compared, this R370S mutation presumably weakened the strong hydrophobic interactions among Cys-368, Leu-413, and Pro-415 (Fig. 3A), thereby partially restoring the protein export activity of the fT3SS with the G368C substitution. This raises a plausible hypothesis that that the conserved GYXLI motif may act as a structural switch to induce the cyclic open-close domain motion of FlhAC.

Hydrophobic parts of the side chain of Tyr-369 and Arg-370 make hydrophobic contacts with Pro-415 and Ala-416 in the open form of wild-type FlhAC, and Ile-372 stabilizes these hydrophobic interactions through the hydrophobic contact with Tyr-369. Leu-371 makes a hydrophobic contact with Pro-434 (Fig. 4A). To clarify the role of the conserved GYXLI motif of FlhAC in flagellar protein export by the fT3SS, we constructed the Y369A/R370A/L371A/I372A (here referred to as AAAA) and Y369G/R370G/L371G/I372G (here referred to as GGGG) mutants and analyzed their motility in 0.35% soft agar. Immunoblotting with polyclonal anti-FlhAC antibody revealed that the AAAA and GGGG mutations did not affect the steady expression level of FlhA (Fig. 4B). The AAAA mutant showed a very weak motility phenotype (Fig. 4C). In agreement with this motility phenotype, more than 80% cells of the AAAA strain produced shorter flagellar filaments than those of wild-type cells (Fig. 4D). The GGGG mutation caused a nonmotile phenotype (Fig. 4C). Consistently, the GGGG mutant did not produce the flagellar filaments at all (Fig. 4D). These observations suggest that a conformational change of the GYXLI motif through remodeling of hydrophobic interaction networks seen in this motif may be critical for efficient flagellar protein export.

FIG 4

Effect of the Y369A/R370A/L371A/I372A and Y369G/R370G/L371G/I372G mutations on flagellar filament formation. (A) Hydrophobic side chain network seen in the conserved GYXLI motif of wild-type FlhAC (PDB ID: 3A5I). (B) Immunoblotting, using polyclonal anti-FlhAC antibody, of whole-cell proteins (Cell) from NH001 (ΔflhA) carrying pTrc99AFF4 (indicated by V), pMM130 (indicated by WT), pMKM130-A4 (indicated by AAAA), and pMKM130-G4 (indicated by GGGG), which were exponentially grown at 30°C with shaking. (C) Motility of the above-described transformants in 0.35% soft agar. Plates were incubated at 30°C for 6.5 h. (D) Fluorescent images of the same transformants. Fresh transformant cells were grown in L-broth containing ampicillin until the cells reached the stationary phase, and then flagellar filaments were labeled with a fluorescent dye, Alexa Fluor 594. The fluorescence images of the filaments labeled with Alexa Fluor 594 (magenta) were merged with the bright-field images of the cell bodies.

Effect of intragenic flhA(M365I), flhA(A446E), and flhA(P550S) suppressor mutations on the hydrophobic side chain interaction networks.

The intragenic flhA(M365I), flhA(A446E), and flhA(P550S) mutations have been reported to restore motility defects in the flhA(G368C) mutant at 42°C (24, 28). To clarify how the flhA(G368C) mutation stabilizes the completely closed form of FlhAC at 42°C through the strong hydrophobic interactions between Gln-498 and Pro-667 and those between Phe-459 and Pro-646, we next analyzed the effect of the M365I, A446E, and P550S suppressor mutations on the conformational changes of FlhAC-G368C.

Pro-550 of domain D3 makes a hydrophobic contact with Met-398 of domain D1 (Fig. 1B), and therefore the P550S substitution should weaken the hydrophobic interactions between domains D1 and D3. Met-365 of domain D1 made a strong hydrophobic contact with Leu-492 of domain D1 in the closed form of FlhAC-G368C but not in its open form (Fig. 3B). Therefore, we assume that the M365I substitution affects this hydrophobic interaction to induce the conformational change of domain D1, thereby weakening the hydrophobic interaction between Gln-498 and Pro-667 in FlhAC-G368C.

It has been shown that the flhA(G368C) mutation induces a conformational change of domain D2 as judged by far-UV circular dichroism (CD) measurements of purified FlhAC with the G368C substitution (29). Ala-446 of domain D2 hydrophobically interacted with Gln-477 of domain D2 in both open and closed forms of FlhAC-G368C (Fig. 3C), so the A446E mutation would induce the formation of a hydrogen bond between Glu-446 and Gln-447 as well as a hydrophobic contact. Therefore, we assume that this A446E mutation may induce a conformational change of domain D2, affecting the hydrophobic contact between Phe-459 and Pro-646.

Mutational analysis of residues forming hydrophobic side chain interaction networks in FlhAC-G368C.

MD simulation of FlhAC-G368C suggests that the flhA(G368C) mutation may induce hydrophobic side chain interaction networks in FlhAC to stabilize a complete closed form at 42°C and that Leu-413, Pro-415, Pro-434, Phe-459, Gln-477, Leu-492, Gln-498, Lys-548, Pro-646, and Pro-667 may be involved in dynamic open-close domain motions of FlhAC (Fig. 1B, 3, and 4A). To test whether the flhA(G368C) mutation affects the hydrophobic side chain interaction networks in FlhAC, we constructed flhA(G368C) mutants with either L413A, P415A, P434A, F459A, Q477A, L492A, Q498A, K548A, P646A, or P667A substitution and analyzed the motility of these double mutants at both 30°C and 42°C (Fig. 5A). These double mutations did not affect the steady expression level of FlhA as judged by immunoblotting with polyclonal anti-FlhAC antibody (Fig. 5B).

FIG 5

Effect of alanine substitution in residues involved in remodeling of hydrophobic side chain interaction networks in FlhAC-G368C on flagellar protein export by the flhA(G368C) mutant. (A) Motility of NH001 (ΔflhA) carrying pTrc99AFF4 (indicated by V), pMM130 (indicated by WT), pYI130(G368C) (indicated by G368C), pMKM130(G368C/L413A) (indicated by G368C/L413A), pMKM130(G368C/P415A) (indicated by G368C/P415A), pMKM130(G368C/P434A) (indicated by G368C/P434A), pMKM130(G368C/F459A) (indicated by G368C/F459A), pMKM130(G368C/Q477A) (indicated by G368C/Q477A), pMKM130(G368C/L492A) (indicated by G368C/L492A), pMKM130(G368C/Q498A) (indicated by G368C/Q498A), pMKM130(G368C/K548A) (indicated by G368C/K548A), pMKM130(G368C/P646A) (indicated by G368C/P646A), or pMKM130(G368C/P667A) (indicated by G368C/P667A) in 0.35% soft agar. Plates were incubated at 30°C for 6.5 h and at 42°C for 6 h. (B) Secretion assays of FlgD and FlgK. Immunoblotting, using polyclonal anti-FlgD (1st row), anti-FlgK (2nd row), or anti-FlhAC (3rd row) antibody, of whole-cell proteins (Cell) and culture supernatants (Sup) prepared from the above-described strains, which were exponentially grown at 30°C with shaking. The positions of FlgD, FlgK, and FlhA are indicated by arrows. Molecular mass markers (kDa) are shown on the left.

The P415A, P434A, Q477A, Q498A, and K548A substitutions reduced the motility of the flhA(G368C) mutant even at 30°C (Fig. 5A). Consistently, these five substitutions reduced the secretion levels of FlgD and FlgK by the fT3SS (Fig. 5B). Furthermore, they considerably reduced the motility of the flhA(G368C) mutant at 42°C (Fig. 5A). The P415A, P434A, Q477A, Q498A, and K548A substitutions themselves displayed no phenotype at both 30°C and 42°C (Fig. 6A). Therefore, we suggest that the flhA(G368C) mutation affects the hydrophobic side chain interaction networks in FlhAC, thereby inducing conformational changes of domains D1 and D2.

FIG 6

Effect of temperature on the protein export function of FlhA containing a single alanine substitution. (A) Motility of NH001 (ΔflhA) carrying pTrc99AFF4 (indicated by V), pMM130 (indicated by WT), pYI130(G368C) (indicated by G368C), pMKM130(L413A) (indicated by L413A), pMKM130(P415A) (indicated by P415A), pMKM130(P434A) (indicated by P434A), pMKM130(F459A) (indicated by F459A), pMKM130(Q477A) (indicated by Q477A), pMKM130(L492A) (indicated by L492A), pMKM130(Q498A) (indicated by Q498A), pMKM130(K548A) (indicated by K548A), pMKM130(P646A) (indicated by P646A), or pMKM130(P667A) (indicated by P667A) in 0.35% soft agar. Plates were incubated at 30°C and 42°C for 5 h. (B) Secretion assays of FlgD and FlgK. Immunoblotting, using polyclonal anti-FlgD (1st row) or anti-FlgK (2nd row) antibody, of whole-cell proteins (Cell) and culture supernatants (Sup) prepared from NH001 carrying pTrc99A, pMM130, pYI130(G368C), pMKM130(F459A), or pMKM130(L492A), which were exponentially grown at either 30°C (left panels) or 42°C (right panels). The positions of FlgD, and FlgK are indicated by arrows. Molecular mass markers (kDa) are shown on the left.

The L413A and L492A substitutions interfered with the motility of the flhA(G368C) mutant at both 30°C and 42°C (Fig. 5A). Consistently, these two substitutions inhibited the secretion of both FlgD and FlgK by the flhA(G368C) mutant grown at 30°C (Fig. 5B). The L413A mutation itself did not inhibit motility at either 30°C or 42°C (Fig. 6A, upper panels). Because Cys-368 makes strong hydrophobic contacts with Arg-370, Leu-413, and Pro-415 of domain D1 in the completely closed form of FlhAC-G368C (Fig. 3A), we suggest that the L413A mutation stabilizes these hydrophobic contacts even at 30°C, thereby inhibiting the protein transport activity of the fT3SS. In contrast to the L413A mutation, the L492A substitution itself significantly reduced motility at 42°C but not at 30°C (Fig. 6A, lower panels). Consistently, this substitution inhibited the secretion of FlgD and FlgK by the fT3SS at 42°C but not at 30°C (Fig. 6B). These results indicate that the temperature shift-up from 30°C to 42°C induces a conformational change of domain D1 of FlhAC by the L492A substitution, thereby reducing the protein export activity of the fT3SS. Because Leu-492 makes a hydrophobic contact with Met-365 in the closed form of FlhAC-G368C but not in its open form (Fig. 3B), we suggest that a proper switching of the hydrophobic side chain interaction between Met-365 and Leu-492 is critical for flagellar protein export by the fT3SS.

Phe-459 of FlhA is located within a highly conserved hydrophobic dimple at the interface between domains D1 and D2 (Fig. 1B) and is directly involved in substrate recognition (16, 17, 23, 24, 26). The F459A mutation reduced the motility of the flhA(G368C) mutant at both 30°C and 42°C (Fig. 5A, upper panels). This mutation reduced the level of FlgK secretion by the flhA(G368C) mutant grown at 30°C but not the level of FlgD secretion (Fig. 5B), indicating that the F459A substitution affects the docking of the FlgN-FlgK chaperone-export substrate complex to FlhAC-G368C but not that of FlgD, in agreement with previous reports (17, 21). Because the motility of the flhA(G368C/F459A) mutant was much worse than that of the flhA(F459A) mutant (Fig. 5 and 6), we suggest that the G368C mutation induces a conformational change of the conserved hydrophobic dimple involved in the interaction with flagellar chaperones. Furthermore, the temperature shift-up from 30°C to 42°C reduced the levels of both FlgD and FlgK secretion by the flhA(F459A) mutant (Fig. 6B), thereby reducing motility at 42°C (Fig. 6A). Therefore, we propose that a remodeling of hydrophobic side chain interaction networks in the conserved hydrophobic dimple may be required for efficient and stable binding of export substrates and chaperone-substrate complexes to FlhAC.

The P646A and P667A mutations, which are located within domain D4, did not affect the motility of the flhA(G368C) mutant at both 30°C and 42°C (Fig. 5A, lower panels). These two mutations themselves showed no phenotype (Fig. 6A, lower panels), indicating that these two proline residues are not critical for the FlhA function. The structural transition from the open conformation to the closed conformation occurs in at least two steps (12, 24). The first conformational change occurs at the interface between domains D3 and D4, resulting in a 13° rotation of domain D4 in the direction toward domain D2, and the second conformational change occurs at a flexible hinge between domains D1 and D3, allowing FlhAC to adopt the closed form. Therefore, we suggest that the flhA(G368C) mutation may limit the hinge movement between domains D1 and D3 through a remodeling of the hydrophobic side chain interaction networks of domain D1, thereby allowing Pro-646 and Pro-667 to make the strong hydrophobic contacts with Phe-459 and Gln-498, respectively (Fig. 1B).

DISCUSSION

The temperature-sensitive flhA(G368C) mutation limits the cyclic open-close domain motions of FlhAC at 42°C, thereby reducing the flagellar protein export by the fT3SS and therefore the cell motility (24). Gly-368 is located within the highly conserved GYXLI motif of FlhAC (Fig. 1A), suggesting that the GYXLI motif may be involved in such domain motions of FlhAC. To clarify the role of Gly-368 in flagellar protein export, we performed mutational analysis of FlhAC-G368C combined with MD simulation and showed that the flhA(G368C) mutation induced a remodeling of hydrophobic side chain interaction networks in FlhAC at 42°C, allowing Gln-498 of domain D1 and Phe-459 of domain D2 to hydrophobically interact with Pro-667 and Pro-646 of domain D4, respectively (Fig. 1B). As a result, the flhA(G368C) mutation not only suppresses the dynamic open-close domain motions of FlhAC but also stabilizes the completely closed conformation at 42°C. Intragenic M365I, R370S, A446E, and P550S suppressor mutations, which restore motility defects in the flhA(G368C) mutant to near wild-type levels (24, 28), affected the hydrophobic side chain interaction networks in the closed FlhAC structure, thereby restoring the protein export activity of the fT3SS containing the flhA(G368C) mutation (Fig. 3). Cys-368 hydrophobically interacts with Arg-370, Leu-413, and Pro-415 of domain D1 in the closed form of FlhAC-G368C but not in its open form (Fig. 3A). Because the AAAA and GGGG mutations reduced motility considerably (Fig. 4C), we propose that the conformational flexibility of FlhAC by Gly-368 may be required for cyclic conformational changes of the conserved GYXLI motif, allowing FlhAC to undergo the cyclic open-close domain motions through the remodeling of the hydrophobic side chain interaction networks in FlhAC.

FlhAC forms a nonameric ring structure in the fT3SS (14, 30). The cryo-electron microscopy (cryo-EM) structure of the SctVC ring derived from the enteropathogenic Escherichia coli T3SS injectisome, which is an FlhAC homolog, has shown that the SctVC subunit adopts a closed conformation and maintained the closed conformation during MD simulation (31). In contrast, the cryo-EM structure of SctVC of the Salmonella SPI-2 T3SS injectisome undergoes dynamic open-closed domain motions in a manner similar to that of FlhAC (32). Here, we showed that the completely closed form of FlhAC reflected an inactive state of the fT3SS and that the cyclic open-close domain motions of FlhAC were critical for efficient and rapid flagellar protein transport by the fT3SS (Fig. 2). The FlgN, FliS, and FliT chaperones in complex with their cognate substrates bind to FlhAC at a nanomolar affinity (17, 33), and such strong interactions between the chaperones and FlhAC assist protein unfolding and export by the fT3SS (34, 35). The flagellar chaperones bind to the open form of FlhAC but not to the closed form (23, 24). Purified FlhAC-G368C prefers to adopt a closed conformation even at room temperature (24). Because pulldown assays by glutathione S-transferase (GST) affinity chromatography have revealed that the flhA(G368C) mutation reduces the binding affinity of FlhAC for the chaperone-substrate complex (24), we propose that the structural transition of FlhAC from the open to the closed form may induce the dissociation of empty chaperone from FlhAC for the binding of a newly delivered chaperone-substrate complex for the export of the next substrate.

FliJ binds to FlhAL with high affinity and opens both the H+ and polypeptide channels of the transmembrane export gate complex, allowing the fT3SS to couple H+ flow through the H+ channel with the translocation of export substrate through the polypeptide channel (Fig. S1B) (36). FliJ also binds to FlhAC with low affinity (26). It has been shown that CdsO, an FliJ homolog, binds to CdsVC, an FlhAC homolog, at a large cleft between the D4 domains of neighboring CdsVC subunits in the CdsVC ring structure (37). This suggests that FliJ also binds to the cleft between the D4 domains of neighboring FlhAC subunits in the FlhAC ring. It has been reported that FliJ stabilizes the interaction between FlhAC and FliT in complex with FliD (15). This raises the possibility that the interaction of FliJ with the cleft between the D4 domains in the FlhAC ring may stabilize the open conformation of FlhAC subunits in the ring. To explore this possibility, we built the models of the FlhAC ring in complex with FliJ in the open and closed forms of FlhAC based on the crystal structure of the CdsVC ring in complex with CdsO (PDB ID: 6WA9) (Fig. 7). FliJ can bind to the cleft between the D4 domains of neighboring FlhAC subunits stably when FlhAC adopts the open form in the ring structure (right panel) because FliJ can contact both FlhAC subunits. However, the FliJ binding to FlhAC in the closed form appears to be unstable (left panel) because the distance between the neighboring D4 domains is longer in the closed form (ca. 80.8 Å) than that in the open form (ca. 36.9 Å), restraining the proper contacts of FliJ with two FlhAC subunits (middle panel). Because the open and closed forms of FlhAC represent the active and inactive states of the fT3SS, respectively (Fig. 2), we propose that the cyclic open-close domain motion of FlhAC may be important for an efficient and robust energy coupling mechanism of the fT3SS.

FIG 7

Model for of the closed (left panel, magenta) and open (right panel, cyan) forms of the FlhAC ring with FliJ. The closed and open ring models were made by fitting domains D1 and D2 of the open form of FlhAC (PDB ID: 3A5I) and its closed form obtained by MD simulation to those of MxiAC in the nonameric ring structure (PDB ID: 4A5P). The CdsO-CdsVC complex structure (PDB ID: 6WA9) was superimposed on the FlhAC ring models, and then FliJ (green) (PDB ID: 3AJW) was superimposed on the 6WA9 structure to build the FlhAC-FliJ ring complex. Two FlhAC subunits in the ring model are shown. FliJ can bind to a cleft between domains D4 of neighboring FlhAC subunits in the open form of the FlhAC ring (right panel). Because a distance between the D4 domains is longer in the closed form than in the open form (center panel), FliJ presumably cannot bind to the cleft in the closed form.

MATERIALS AND METHODS

Bacterial strains, P22-mediated transduction, and DNA manipulations.

The Salmonella strains used in this study are listed in Table 1. P22-mediated transductional crosses were performed with P22HTint (38). DNA manipulations were performed using standard protocols. Site-directed mutagenesis was carried out using Prime STAR Max premix as described in the manufacturer’s instructions (TaKaRa Bio). All of the flhA mutations were confirmed by DNA sequencing (Eurofins Genomics).

TABLE 1

Strains and plasmids used in this study

Strain/plasmid	Relevant characteristics	Reference or source
Salmonella strain
SJW1103	Wild-type for motility and chemotaxis	46
SJW2228	flhA(G368C)	27
NH001	ΔflhA	47
Plasmids
pTrc99AFF4	Expression vector	48
pMM130	pTrc99AFF4/FlhA	49
pYI130(G368C)	pTrc99AFF4/FlhA(G368C)	24
pMKM130-A4	pTrc99AFF4/FlhA(T369A/R370A/L371A/I372A)	This study
pMKM130-G4	pTrc99AFF4/FlhA(T369G/R370G/L371G/I372G)	This study
pMKM130(L413A)	pTrc99AFF4/FlhA(L413A)	This study
pMKM130(G368C/L413A)	pTrc99AFF4/FlhA(G368C/L413A)	This study
pMKM130(P415A)	pTrc99AFF4/FlhA(L415A)	This study
pMKM130(G368C/P415A)	pTrc99AFF4/FlhA(G368C/P415A)	This study
pMKM130(P434A)	pTrc99AFF4/FlhA(P434A)	This study
pMKM130(G368C/P434A)	pTrc99AFF4/FlhA(G368C/P434A)	This study
pMKM130(F459A)	pTrc99AFF4/FlhA(F459A)	This study
pMKM130(G368C/F459A)	pTrc99AFF4/FlhA(G368C/F459A)	This study
pMKM130(Q477A)	pTrc99AFF4/FlhA(Q477A)	This study
pMKM130(G368C/Q477A)	pTrc99AFF4/FlhA(G368C/Q477A)	This study
pMKM130(L492A)	pTrc99AFF4/FlhA(L492A)	This study
pMKM130(G368C/L492A)	pTrc99AFF4/FlhA(G368C/L492A)	This study
pMKM130(Q498A)	pTrc99AFF4/FlhA(Q498A)	This study
pMKM130(G368C/Q498A)	pTrc99AFF4/FlhA(G368C/Q498A)	This study
pMKM130(K548A)	pTrc99AFF4/FlhA(K548A)	This study
pMKM130(G368C/K548A)	pTrc99AFF4/FlhA(G368C/K548A)	This study
pMKM130(P646A)	pTrc99AFF4/FlhA(P646A)	This study
pMKM130(G368C/P646A)	pTrc99AFF4/FlhA(G368C/P646A)	This study
pMKM130(P667A)	pTrc99AFF4/FlhA(P667A)	This study
pMKM130(G368C/P667A)	pTrc99AFF4/FlhA(G368C/P667A)	This study

Motility assay.

Fresh colonies were inoculated onto soft agar plates (1% [wt/vol] tryptone, 0.5% [wt/vol] NaCl, 0.35% [wt/vol] Bacto agar) containing 50 μg/mL ampicillin, and then the plates were incubated at 30°C. At least six measurements were performed.

Observation of flagellar filaments with a fluorescent dye.

Salmonella cells were grown at 30°C in 5 mL of L-broth (1% [wt/vol] Bacto-tryptone, 0.5% [wt/vol] Bacto-yeast extract, 0.5% [wt/vol] NaCl) containing 100 μg/mL ampicillin. The cells were attached to a coverslip (Matsunami Glass, Japan), and unattached cells were washed away with motility buffer (10 mM potassium phosphate, pH 7.0, 0.1 mM EDTA, 10 mM l-sodium lactate). A 1-μL aliquot of polyclonal anti-FliC serum was mixed with 50 μL of motility buffer, and then 50 μL of the mixture was applied to the cells attached to the cover slip. After washing with the motility buffer, 1 μL of anti-rabbit IgG conjugated with Alexa Fluor 594 (Invitrogen) was added to 50 μL of motility medium, and then the mixture was applied. After washing with the motility buffer, the cells were observed by fluorescence microscopy (39). Fluorescence images were analyzed using ImageJ software version 1.52 (National Institutes of Health).

Secretion assay.

Salmonella cells were grown in 5 mL of L-broth containing 100 μg/mL ampicillin at either 30°C or 42°C with shaking until the cell density reached an optical density at 600 nm (OD600) of ca. 1.2 to 1.4. Cultures were centrifuged to obtain cell pellets and culture supernatants. The cell pellets were resuspended in sodium dodecyl sulfate (SDS)-loading buffer solution (62.5 mM Tris-HCl, pH 6.8, 2% [wt/vol] SDS, 10% [wt/vol] glycerol, 0.001% [wt/vol] bromophenol blue) containing 1 μL of 2-mercaptoethanol. Proteins in the culture supernatants were precipitated by 10% trichloroacetic acid and suspended in a Tris/SDS loading buffer (1 volume of 1 M Tris, 9 volumes of 1× SDS-loading buffer solution) containing 1 μL of 2-mercaptoethanol (40). Both whole cellular proteins and culture supernatants were normalized to a cell density of each culture to give a constant number of Salmonella cells. After proteins were boiled in both whole cellular and culture supernatant fractions at 95°C for 3 min, these protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Cytiva) using a transblotting apparatus (Hoefer). Then, immunoblotting with polyclonal anti-FlgD, anti-FlgK, or anti-FlhAC antibody was carried out using an iBind Flex Western device (Thermo Fisher Scientific) as described in the manufacturer’s instructions. Detection was performed with Amersham ECL Prime Western blotting detection reagent (Cytiva). Chemiluminescence signals were captured by a Luminoimage LAS-3000 analyzer (FujiFilm). At least three measurements were performed.

Multiple sequence alignment.

Multiple sequence alignment was carried out using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/).

MD simulation.

MD simulations of FlhAC with either G368C/K548C or G368C/F459C/K548C substitution were conducted as described previously (24). The mutant structures were constructed based on the 3A5I structure of FlhAC (12). The Amber ff14SB force field (41) was used for the proteins. Each mutant was initially solvated in a cubic box of SPC/Eb water molecules (42) and 0.17 M KCl (43) with a margin of at least 12 Å from the proteins to the periodic box boundaries. The simulation was conducted with the pmemd.cuda module (44) of AMBER20/21 (45). MD simulations of the G368C/K548C and G368C/F459C/K548C mutants were independently conducted at 300 K. After energy minimization and equilibration MD for 1 ns with positional restraints for the protein main chain atoms at 300 K and 1 atm, MD simulation without restraints was conducted for 1.5 μs.