Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus

The switch in export specificity of the type Ill flagellar protein export apparatus from rod/hook type to filament type is believed to occur upon completion of hook assembly by way of an interaction of the type Ill secretion substrate specificity switch (T3S4) domain of the hook-length control protein FliK, with the integral membrane export apparatus component FIhB. The T3S4 domain of FliK (FliK(T3S4)) consisting of an-Lino acid residues 265-405 has an unstable and flexible conformation in its last 35 residues (FliK(CT)). To investigate the role of FliK(T3S4) in substrate specificity switching, we studied the effect of deletions and point mutations within this domain and characterized suppressor mutations. Deletions of ten amino acid residues within the region of residues 301-350 and five amino acids of residues 401-405 abolished switching of export specificity. Site directed mutagenesis showed that highly conserved residues, Va1302, 11604, Leu335, Va1401 and Ala405, are essential, and that the five C terminal residues (401405) are restricted in conformation for the switching process. Suppressor mutant analysis of the fliK(S319Y) mutant, which produces extended hooks with filaments attached due to delayed switching, suggested that FliK(T3S4) interacts with the C terminal half of the cytoplasmic domain of FIhB (FlhB(C)). We propose a two step binding model of FliK(T3S4) and FlhB(C), in which residues 301-350 of FliK bind to FlhB(C) upon hook assembly completion at about 55 nm, and then unfolded FliK(CT) binds to FlhB(C) to trigger the switch in substrate specificity. (c) 2006 Elsevier Ltd. All rights reserved.