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

FliP, FliQ, and FliR forms a membrane-embedded channel for flagellar protein export. How these subunits assemble into the functional channel remains unclear. Here we combined topological mapping, cryo-EM image analysis, and biochemical assays to dissect the assembly mechanism of the FliPQR complex. Using PhoA fusion assays, we show that membrane-spanning helices of each subunit are repositioned toward the periplasm upon complex assembly, revealing large-scale topological remodeling. FliP alone exists in a dynamic equilibrium between pentameric and hexameric states, indicating that the sixth FliP subunit associates weakly and dissociates easily. FliR inserts into the gap between the first and fifth FliP subunits, forming a stable FliPR complex. The FliQ-like element in FliR efficiently recruits the first FliQ subunit. These findings uncover a previously unrecognized mechanism by which hierarchical assembly and structural remodeling drive the efficient and robust formation of the flagellar export channel.