Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus

Bacteria use a type III protein export apparatus for construction of the flagellum, which consists of the basal body, the hook, and the filament. FlhA forms a homo-nonamer through its C-terminal cytoplasmic domains (FlhA_C) and ensures the strict order of flagellar assembly. FlhA_C goes through dynamic domain motions during protein export, but it remains unknown how it occurs. Here, we report that the FlhA(G368C) mutation biases FlhA_C toward a closed form, thereby reducing the binding affinity of FlhA_C for flagellar export chaperones in complex with their cognate filament-type substrates. The G368C mutations also restrict the conformational flexibility of a linker region of FlhA (FlhA_L), suppressing FlhA_C ring formation. We propose that interactions of FlhA_L with its neighboring subunit converts FlhA_C in the ring from a closed conformation to an open one, allowing the chaperon/substrate complexes to bind to the FlhA_C ring to form the filament at the hook tip.