Many bacteria utilize flagella to swim in viscous liquids and move around on solid surfaces to migrate towards more favorable environments for their survival. The flagellum is a supramolecular complex consisting of the basal body, which acts as a rotary motor, the filament, which functions as a helical propeller and the hook, which connects the basal body and filament and works as a universal joint to smoothly transmit torque produced by the motor to the filament 1.
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 building blocks from the cytoplasm to the distal end of the growing structure. The fT3SS consists of five transmembrane proteins, FlhA, FlhB, FliP, FliQ and FliR, and three cytoplasmic proteins, FliH, FliI and FliJ (Fig. 1a)2.
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 flagellar base (Fig. 1a)6. An interaction between FliH and a C ring protein, FliN, is required for efficient localization of the ATPase ring complex to the flagellar base7,8. 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 manner9. 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 source10,11.
FlhA acts as an export engine fueled by both PMF and SMF10. An interaction between FliJ and the C-terminal cytoplasmic domain of FlhA (FlhAC) activates the protein export channel to couple either H+ or Na+ flow with the translocation of flagellar building blocks across the cytoplasmic membrane5,10,12. FlhAC forms a homo-nonamer in the fT3SS13,14 and serves as a docking platform that brings the order in the export substrates for efficient flagellar assembly15–18.
FlhAC consists of four compactly folded domains, D1, D2, D3, and D4, and a flexible linker (FlhAL) connecting FlhAC and FlhATM (Fig. 1b)19. FlhAC 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 form21,22. FlhAL stabilizes the open form, allowing the chaperones in complex with their cognate substrates to efficiently bind to the FlhAC ring to promote filament assembly at the hook tip14,22. Interestingly, FlhAL also binds to the chaperone-binding site of the open form during hook assembly, thereby not only suppressing premature docking of the chaperones to FlhAC but also facilitating the export of the hook protein23. These observations suggest that the open form of FlhAC reflect 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 at a restrictive temperature of 42ºC but not at a permissive temperature of 30ºC22,24−26. The temperature shift-up from 30ºC to 42ºC immediately arrests the export of flagellar building blocks, suggesting that this induces a conformational change of FlhAC24,26,27. Molecular dynamics (MD) simulation has shown that the flhA(G368C) mutation restricts dynamic open-close domain motions of FlhAC at 42ºC, thereby stabilizing a completely closed form of FlhAC22. Interestingly, the flhA(G368C/K548C) mutation results in a loss-of-function phenotype even at 30ºC22. However, it remains unknown why this mutation interferes with flagellar protein export at 30ºC. To clarify this, we carried out genetic analysis of the flhA(G368C/K548C) mutant. We provide evidence that the FlhA(G368C/K548C) mutation stabilizes hydrophobic side-chain interactions between domains D1 and D3 and between D2 and D4, thereby suppressing dynamic open-close domain motions of FlhAC, and that suppressor mutations in FlhAC induces remodeling in the hydrophobic interaction networks in FlhAC, allowing FlhAC with the G368C/K548C mutation to restore the cyclic open-close domain motions.