FlhA Undergoes Cyclic Open-close Domain Motions During Flagellar Protein Export in Salmonella

The agellar type III secretion system (fT3SS) transports agellar building blocks from the cytoplasm to the distal end of the growing agellar structure. The C-terminal cytoplasmic domain of FlhA (FlhA C ) serves as a docking platform for agellar chaperones in complex with their cognate substrates and ensures the strict order of protein export for ecient agellar assembly. FlhA C adopts open and closed conformations, and the chaperones bind to the open form, allowing the fT3SS to transport the substrates to the cell exterior. To clarify the role of the closed form in agellar protein export, we isolated pseudorevertants from the hA(G368C/K549C) mutant, in which the closed conformation is stabilized to inhibit the protein transport activity of the fT3SS. Each of M365I, R370S, A446E and P550S substitutions in FlhA C identied in the pseudorevertants affected hydrophobic side-chain interaction networks in the closed FlhA C structure, thereby restoring the protein transport activity to a considerable degree. We propose that a cyclic open-close domain motion of FlhA C is required for rapid and ecient agellar protein export where a structural transition from the open to the closed form induces the dissociation of empty chaperones from FlhA C .


Introduction
Many bacteria utilize agella to swim in viscous liquids and move around on solid surfaces to migrate towards more favorable environments for their survival. The agellum is a supramolecular complex consisting of the basal body, which acts as a rotary motor, the lament, which functions as a helical propeller and the hook, which connects the basal body and lament and works as a universal joint to smoothly transmit torque produced by the motor to the lament 1 .
Flagellar assembly begins with the basal body, followed by the hook and nally the lament. To construct the agellum on the cell surface, the agellar type III secretion system (fT3SS) transports agellar building blocks from the cytoplasm to the distal end of the growing structure. The fT3SS consists of ve transmembrane proteins, FlhA, FlhB, FliP, FliQ and FliR, and three cytoplasmic proteins, FliH, FliI and FliJ FlhA, FlhB, FliP, FliQ and FliR assemble into a protein export channel inside the basal body MS ring formed by the transmembrane protein, FliF (Fig. 1a) 3 . The protein export channel is powered by the transmembrane electrochemical gradient of protons (H + ), namely proton motive force (PMF) 4,5 . FliH, FliI and FliJ forms a cytoplasmic ATPase ring complex at the agellar base (Fig. 1a) 6 . An interaction between FliH and a C ring protein, FliN, is required for e cient localization of the ATPase ring complex to the agellar base 7,8 . ATP hydrolysis by the ATPase ring complex induces gate opening of the protein export channel for the translocation of export substrates across the cytoplasmic membrane in a PMF-dependent manner 9 . However, when the ATPase ring complex does not work properly, the protein channel complex utilizes sodium motive force (SMF) across the cytoplasmic membrane as the energy source 10,11 .
FlhA acts as an export engine fueled by both PMF and SMF 10 . An interaction between FliJ and the Cterminal cytoplasmic domain of FlhA (FlhA C ) activates the protein export channel to couple either H + or Na + ow with the translocation of agellar building blocks across the cytoplasmic membrane 5,10,12 .
FlhA C forms a homo-nonamer in the fT3SS 13,14 and serves as a docking platform that brings the order in the export substrates for e cient agellar assembly [15][16][17][18] .
FlhA C consists of four compactly folded domains, D1, D2, D3, and D4, and a exible linker (FlhA L ) connecting FlhA C and FlhA TM (Fig. 1b) 19 . FlhA C adopts open and closed conformations (Fig. 2a) 19,20 . The FliS/FliC and FliT/FliD chaperone/substrate complexes bind to the chaperone-binding site of the open form but not to that of the closed form 21,22 . FlhA L stabilizes the open form, allowing the chaperones in complex with their cognate substrates to e ciently bind to the FlhA C ring to promote lament assembly at the hook tip 14,22 . Interestingly, FlhA L also binds to the chaperone-binding site of the open form during hook assembly, thereby not only suppressing premature docking of the chaperones to FlhA C but also facilitating the export of the hook protein 23 . These observations suggest that the open form of FlhA C re ect an active state of the fT3SS. However, little is known about the role of the closed form of FlhA C in agellar protein export.
The hA(G368C) mutation inhibits the protein transport activity at a restrictive temperature of 42ºC but not at a permissive temperature of 30ºC 22,24−26 . The temperature shift-up from 30ºC to 42ºC immediately arrests the export of agellar building blocks, suggesting that this induces a conformational change of

Methods
Bacterial strains, P22-mediated transduction and DNA manipulations. Salmonella strains used in this study are listed in Table 1. P22-mediated transductional crosses were performed with P22HTint. DNA manipulations were performed using standard protocols. DNA sequencing reactions were carried out using BigDye v3.1 (Applied Biosystems) and then the reaction mixtures were analyzed by a 3130 Genetic Analyzer (Applied Biosystems).  Isolation of pseudorevertants from the hA(G368C/K548C) mutant. To clarify why the FlhA(G368C/K548C) mutation inhibits agellar protein export, pseudorevertants were isolated from the hA(G368C/K548C) mutant by streaking an overnight culture out on 0.35% soft agar plates, incubating them at 30ºC for a few days and looking for motility halos emerging from the streak. In total ve motile colonies were puri ed from such halos. The motility of these pseudorevertants was better than that of the parent strain at 30ºC although not as good as the wild-type strain (Fig. 2b). In agreement with this, the secretion levels of agellar building blocks such as FlgD, FlgE, FlgK, FlgL and FliC by these pseudorevertants were recovered although not at the wild-type levels (Fig. 2c). P22-mediated transduction showed that all suppressor mutations were co-transduced with the hA(G368C/K548C) mutation, indicating that they are located in the hBAE operon. DNA sequencing revealed that they were all missense mutations in FlhA: M365I, R370S (isolated twice), A446E, and P550S (Fig. 2a) (Fig. 2a). Consistently, this Cys residue is not exposed to the solvent of the molecular surface of FlhA C−G368C as judged by cysteine modi cation with methoxypolyethylene glycol 5000 maleimide 22 . A temperature shift-up from 30ºC to 42ºC remodels these hydrophobic interaction networks in FlhA C−G368C to induce large conformational changes of domains D1 and D2 to get close to domains D3 and D4, respectively (Fig. 3), thereby not only stabilizing a completely closed form but also inhibiting open-close domain motions. The R370S substitution weakens the hydrophobic interactions among Cys-368, Leu-413 and Pro-415 (Fig. 2a) (Fig. 2a).
Therefore the M365I substitution must affect this hydrophobic interaction to induce the conformational change of domain D1 domain (Fig. 3), thereby weakening the hydrophobic interaction between Gln-498 and Pro-667 in FlhA C−G368C . Ala-446 of domain D2 hydrophobically interacts with Gln-477 of domain D2 in the closed form of FlhA C−G368C but not in the open form (Fig. 2a), and the A446E mutation seems to affect this hydrophobic interaction to induce the conformational change of domain D2 (Fig. 3), thereby affecting the hydrophobic contact between Phe-459 and Pro-646. Pro-550 of domain D3 makes a hydrophobic contact with Met-398 of domain D1 (Fig. 2a), and the P550S substitution weakens the hydrophobic contact between domains D1 and D3. Therefore, we propose that the remodeling of the hydrophobic interaction networks in FlhA C−G368C is required for its dynamic open-close domain motions.
Because the G368C mutation is located at the N-terminal end of a hinge loop consisting of residues 368-381, we propose that the conformational exibility of this hinge loop is required for e cient remodeling of the hydrophobic interaction networks in FlhA C .

Discussion
The chaperone-binding site is located at an interface between domains D1 and D2 of FlhA C (Fig. 1b)  FlhA C forms a nonameric ring structure in the fT3SS 13,14 . FliJ binds to FlhA L to activate the fT3SS to drive agellar protein export in a PMF-dependent manner (Fig. 1b)  FlhAC (PDB ID: 3A5I) consists of four compactly folded domains, D1, D2, D3 and D4, and a exible linker region (FlhAL) connecting FlhATM and FlhAC. The Cα backbone is color-coded from blue to red, going through the rainbow colors from the N-to the C-terminus. FliJ binds to FlhAL to active the FlhA ion channel. Flagellar export chaperones (FlgN, FliS, FliT) bind to a well conserved hydrophobic dimple located at the interface between domains D1 and D2.