Dynamic topological remodeling underlies assembly of the flagellar protein-export channel FliPQR complex

FliP, FliQ, and FliR form the membrane-embedded protein-export channel that powers bacterial flagellar assembly. How they assemble into a functional export channel remains unclear. Here we combine topological mapping, cryo-EM image analysis, and biochemical assays to investigate the assembly mechanism of the FliPQR complex. PhoA fusion assays suggest that membrane-spanning helices of each subunit become repositioned toward the periplasm upon complex assembly, supporting substantial topological remodeling. FliP alone forms higher-order oligomers, with pentameric species predominating and putative hexameric species appearing less stable. FliR associates with the FliP assembly to form a stable FliPR complex. The FliQ-like structural element in FliR contributes to recruitment of the first FliQ subunit and promotes stable FliQ assembly. Together, these findings support a working model in which hierarchical assembly of the FliPQR complex is accompanied by substantial topological remodeling, providing a framework for understanding export channel biogenesis in flagellar and related type III secretion systems. Cryo-EM, topology mapping and genetic analyses reveal dynamic topological remodeling during assembly of the bacterial flagellar protein-export channel complex, providing a mechanistic framework for export gate biogenesis.