Abstract
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 (FlhA(C)) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhA(C) 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 FlhA(C). 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 FlhA(C) and promote the cyclic open-close domain motions of FlhA(C). 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 FlhA(C) 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 FlhA(C) 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 (FlhA(C)), a transmembrane subunit of the fT3SS, provides binding sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhA(C) undergoes cyclic open-close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhA(C). However, it remains unknown how it works. Here, we carried out mutational analysis of FlhA(C) combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhA(C) by Gly-368 is important for remodeling hydrophobic side chain interaction networks in FlhA(C) to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly.