Structural basis for substrate specificity switching revealed by the FlhA(A489E) suppressor mutation

The flagellar type III secretion system (fT3SS) switches its substrate specificity from hook-type to filament-type upon completion of hook assembly. This process is regulated by the coordinated actions of FliK, FlhB, and FlhA, but the structural mechanism underlying substrate specificity switching remains unclear. Here, we determined the crystal structures of the C-terminal cytoplasmic domain of FlhA(FlhA_C) carrying the A489E substitution, which partially restores motility of the cleavage-defective flhB(P270A) mutant. The structures were solved in two distinct conformational states: an open form at 3.28 Å resolution and a semi-closed form at 2.90 Å resolution. In parallel, AlphaFold3-based structural predictions suggested that the flexible C-terminal cytoplasmic tail of FlhB (FlhB_CCT) binds to a groove formed between the α2 helix and β2 strand of FlhA_C, overlapping with the intersubunit linker-binding site within the FlhA_C ring. Structural analyses further revealed that the A489E substitution rearranges a local salt-bridge network involving Arg-386 and Glu-483, leading to subtle positional shifts of the α2 helix and remodeling of the predicted FlhB_CCT-binding surface. These structural changes may alter the predicted interaction between FlhA_C and FlhB_CCT, thereby lowering the energetic barrier associated with conformational remodeling of the FlhA_C ring during substrate specificity switching. Together, our results provide structural insight into how local electrostatic remodeling modulates conformational dynamics of FlhA_C and regulates substrate specificity switching in the fT3SS.