Abstract
The bacterial flagellum contains its own type III secretion apparatus that coordinates protein export with assembly at the distal end. While many interactions among export apparatus proteins have been reported, few have been examined with respect to the differential affinities and dynamic relationships that must govern the mechanism of export. FlhB, an integral membrane protein, plays critical roles in both export and the substrate specificity switching that occurs upon hook completion. Reported herein is the quantitative characterization of interactions between the cytoplasmic domain of FlhB (FlhBC) and other export apparatus proteins including FliK, FlhAC and FliI. FliK and FlhAC bound with micromolar affinity. KD for FliI binding in the absence of ATP was 84 nM. ATP-induced oligomerization of FliI induced kinetic changes, stimulating fast-on, fast-off binding and lowering affinity. Full length FlhB purified under solubilizing, nondenaturing conditions formed a stable dimer via its transmembrane domain and stably bound FliH. Together, the present results support the previously hypothesized central role of FlhB and elucidate the dynamics of protein-protein interactions in type III secretion.
Highlights
The bacterial flagellum is a proton-driven rotary nanomachine responsible for motility in many species [1,2,3]
To examine dynamic interactions between FlhBC and other apparatus proteins, FlhBC was used as analyte versus each of the other apparatus proteins as ligand (Fig 1A)
Observed binding was complex and nonspecific binding (NSB) as evidenced by response to BSA as ligand was in many cases significant
Summary
The bacterial flagellum is a proton-driven rotary nanomachine responsible for motility in many species [1,2,3]. Most proteins that comprise a flagellum reside beyond the cytoplasmic membrane and must be secreted. Secretion occurs via a specialized type III secretion system (T3SS or “export apparatus”[4,5]). Utilizing protonmotive force [6,7,8], the apparatus. Characterization of Binding Interactions of FlhB translocates flagellar proteins across the cytoplasmic membrane into the central channel within the growing flagellum through which they transit to their final location [9]. Homologous T3SSs effect many modes of bacterial pathogenesis using needle-like structures that closely resemble flagella [10]
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