The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

Abstract

The bacterial flagellar export switching machinery consists of a ruler protein, FliK, and an export switch protein, FlhB and switches substrate specificity of the flagellar type III export apparatus upon completion of hook assembly. An interaction between the C-terminal domain of FliK (FliK ) and the C-terminal cytoplasmic domain of FlhB (FlhB ) is postulated to be responsible for this switch. FliK has a compactly folded domain termed FliK (residues 268-352) and an intrinsically disordered region composed of the last 53 residues, FliK (residues 353-405). Residues 301-350 of FliK and the last five residues of FliK are critical for the switching function of FliK. FliK is postulated to regulate the interaction of FliK with FlhB , but it remains unknown how. Here we report the role of FliK in the export switching mechanism. Systematic deletion analyses of FliK revealed that residues of 351-370 are responsible for efficient switching of substrate specificity of the export apparatus. Suppressor mutant analyses showed that FliK coordinates FliK action with the switching. Site-directed photo-cross-linking experiments showed that Val-302 and Ile-304 in the hydrophobic core of FliK bind to FlhB . We propose that FliK may induce conformational rearrangements of FliK to bind to FlhB .

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

“…The C‐terminal domain of a FliK homologue, YscP, of the Yersinia injectisome binds to the C‐terminal cytoplasmic domain of a FlhB homologue, YscU, to promote substrate specificity switching of the virulence‐associated type III protein export apparatus (Ho et al, ). Consistently, photo‐cross‐linking experiments have showed a direct interaction between FlhB C and the C‐terminal domain of FliK (FliK C ; Kinoshita, Aizawa, Inoue, Namba, & Minamino, ).…”

“…Extragenic fliK suppressor mutations have been identified in FlhB CC but not in FlhB CN (Kinoshita et al, ; Kutsukake et al, ; Minamino, Ferris, Morioya, Kihara, & Namba, ; Williams et al, ), suggesting that FlhB CC is involved in the substrate specificity switching. Here, to clarify the role of the conserved residues of FlhB CC , Pro‐287, Pro‐311, Leu‐318, Ala‐319, Arg‐320, Leu‐322, Glu‐342 and Leu‐344, in the substrate specificity switching, we carried out mutational analysis of these residues.…”

In Vitro Autonomous Construction of the Flagellar Axial Structure in Inverted Membrane Vesicles

“…When the hook length is too short, FliK is secreted out into the extracellular media. When the hook length reaches approximately 55 nm, the C-terminal domain of FliK binds to FlhB, one of the export gate component proteins, to induce conformational changes of FlhB and FlhA to switch the substrate specificity of the export apparatus [17][18][19][20][21][22]. The filament-type proteins form a complex with their specific cognate chaperones, which prevent premature aggregation and/or proteolysis of their cognates and help them associate with the flagellar protein export apparatus in the cytoplasm [23][24][25].…”

The flexible linker of the secreted FliK ruler is required for export switching of the flagellar protein export apparatus

“…Insertions and deletions in FliK N make the hook longer and shorter, respectively, suggesting that FliK N is a molecular ruler 42 . FliK N binds to FlgD and FlgE [43][44][45] , and deletion of residues 129-159 in FliK N reduces the binding affinity of FliK for FlgE, thereby producing longer hooks with the filament attached. This suggests that the interaction of FliK N with FlgE is required for proper hook length measurement 44 .…”

“…1a) 40,46 . The core domain of FliK C binds to FlhB C to allow the flagellar protein export apparatus to switch its substrate specificity from rod-(FlgB, FlgC, FlgF, FlgG, FlgJ, FliE) and hook-type (FlgD, FlgE, FliK) to filament-type (FlgK, FlgL, FlgM, FliC, FliD) 11,12,28,45,47 . Genetic analyses of FliK CT have suggested that FliK CT controls the export switching activity of FliK C during hook assembly 45,47 .…”