Interactions of bacterial flagellar chaperone–substrate complexes with FlhA contribute to co‐ordinating assembly of the flagellar filament

Abstract

SummaryAssembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C-terminal cytoplasmic domain of FlhA (FlhAC), a membrane component of the export apparatus, provides a binding-site for these chaperone-substrate complexes but it remains unknown how it co-ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhAC is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhAC for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14-fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhAC for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.

Recognition and targeting mechanisms by chaperones in flagellum assembly and operation

“…NMR shows that free FliT does not bind to FlhA C (Fig. S7), in agreement with surface plasmon resonance (SPR) data (36). In contrast, the FliT−FliD C complex binds strongly to FlhA C (Fig.…”

“…FliT was shown by biochemical assays to deliver FliD to the export gate by interacting with the cytoplasmic domain of FlhA (FlhA C ) (11,36). To understand the targeting process, we characterized the interaction of FliT and FliD with FlhA C .…”

“…The N terminus of FliS serves as the FlhAbinding site, and in all three chaperones a highly conserved Tyr residue appears to be essential for efficient FlhA binding (Fig. 6) (36). Thus, the three flagellar chaperones may use similar strategies for substrate binding and activation of the complexes for binding to FlhA and thus for targeting to the export gate.…”

“…FliT is a key chaperone in the assembly and operation of the flagellum. FliT interacts with several flagellar proteins, including the exported substrate FliD and the export apparatus components FliI, FliJ, and FlhA, and negatively regulates transcription of flagellar genes by inhibiting the formation of a DNA complex with the master regulator FlhDC (11,24,28,(32)(33)(34)(35)(36). FliD is exported first during the filament assembly to form a pentameric cap that promotes selfassembly of FliC (37).…”

Crystallization and preliminary X-ray analysis of the periplasmic domain of FliP, an integral membrane component of the bacterial flagellar type III protein-export apparatus

“…The C-terminal cytoplasmic domain of FlhA (FlhA C ) has been visualized at the centre of the cavity within the C ring (Abrusci et al, 2013;Kawamoto et al, 2013). FlhA C and the C-terminal cytoplasmic domain of FlhB (FlhB C ) provide binding sites for the ATPase complex, flagellar chaperones and export substrates (Bange et al, 2010;Kinoshita et al, 2013;Minamino & Macnab, 2000;Minamino et al, 2003. Both FlhA C and FlhB C act as an export switch to coordinate flagellar protein export with assembly (Hirano et al, 2009;Kinoshita et al, 2013).…”