Mutational analysis of the C-terminal cytoplasmic domain of FlhB, a transmembrane component of the flagellar type III protein export apparatus in Salmonella

摘要

The flagellar protein export apparatus switches its substrate specificity when hook length has reached approximately 55 nm in Salmonella. The C-terminal cytoplasmic domain of FlhB (FlhB(C)) is involved in this switching process. FlhB(C) consists of FlhB(CN) and FlhB(CC) polypeptides. FlhB(CC) has a flexible C-terminal tail (FlhB(CCT)). FlhB(CC) is involved in substrate recognition, and conformational rearrangements of FlhB(CN)-FlhB(CC) boundary are postulated to be required for the export switching. However, it remains unknown how it occurs. To clarify this question, we carried out mutational analysis of highly conserved residues in FlhB(C). The flhB(E230A) mutation reduced the FlhB function. The flhB(E11S) mutation restored the protein transport activity of the flhB(E230A) mutant to the wild-type level, suggesting that the interaction of FlhB(CN) with the extreme N-terminal region of FlhB is required for flagellar protein export. The flhB(R320A) mutation affected hydrophobic interaction networks in FlhB(CC), thereby increasing insolubility of FlhB(C). The R320A mutation also affected the export switching, thereby producing longer hooks with the filament attached. C-terminal truncations of FlhB(CCT) induced a conformational change of FlhB(CN)-FlhB(CC) boundary, resulting in a loose hook length control. We propose that FlhB(CCT) may control conformational arrangements of FlhB(CN)-FlhB(CC) boundary through the hydrophobic interaction networks of FlhB(CC).