FlhA Undergoes Cyclic Open-close Domain Motions During Flagellar Protein Export in Salmonella
Abstract The flagellar type III secretion system (fT3SS) transports flagellar building blocks from the cytoplasm to the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAC) serves as a docking platform for flagellar chaperones in complex with their cognate substrates and ensures the strict order of protein export for efficient flagellar assembly. FlhAC 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 flagellar protein export, we isolated pseudorevertants from the flhA(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 FlhAC identified in the pseudorevertants affected hydrophobic side-chain interaction networks in the closed FlhAC structure, thereby restoring the protein transport activity to a considerable degree. We propose that a cyclic open-close domain motion of FlhAC is required for rapid and efficient flagellar protein export where a structural transition from the open to the closed form induces the dissociation of empty chaperones from FlhAC.