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Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics.

McMurry JL, Minamino T, Furukawa Y, Francis JW, Hill SA, Helms KA, Namba K - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Cellular Biology, Kennesaw State University, Kennesaw, Georgia, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

FlhBC kinetic survey.Purified T3S proteins were biotinylated and used as ligands. All analyte concentrations were 1 μM. Association and dissociation phases were 180 seconds in all cases. A, FlhBC analyte binding to ligands FlhAC (red), FlhBC (blue), FliH (green), FliI (orange), FliJ (magenta) and FliK (black). B, nonspecific binding control of analyte apparatus proteins binding to sensor-tethered biotinylated BSA. Analyte colors are the same as ligand colors in A. C, Analyte apparatus proteins binding to ligand FlhBC. Colors are the same as in B.
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pone.0134884.g001: FlhBC kinetic survey.Purified T3S proteins were biotinylated and used as ligands. All analyte concentrations were 1 μM. Association and dissociation phases were 180 seconds in all cases. A, FlhBC analyte binding to ligands FlhAC (red), FlhBC (blue), FliH (green), FliI (orange), FliJ (magenta) and FliK (black). B, nonspecific binding control of analyte apparatus proteins binding to sensor-tethered biotinylated BSA. Analyte colors are the same as ligand colors in A. C, Analyte apparatus proteins binding to ligand FlhBC. Colors are the same as in B.

Mentions: 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). A starkly different shift profile from nonspecific control binding (Fig 1B, black trace) was observed for FliK. Smaller differences were noted for all other export proteins, e.g. a small amplitude fast on state for FlhAC, indicating some interaction with FlhBC. Observed binding was complex and nonspecific binding (NSB) as evidenced by response to BSA as ligand was in many cases significant. FliJ, FliH and FlhBC ligands also exhibited binding different from BSA, but were resistant to further analysis due to NSB, low signal and other reasons. Biotinylated FlhBC was tethered to SA sensors and screened for binding versus analyte soluble export apparatus proteins at 1 μM (Fig 1C). Differences in FliI binding were noted. FliK-FlhBC, FlhAC-FlhBC and FlhBC-FliI interactions were selected for further kinetic characterization.


Weak Interactions between Salmonella enterica FlhB and Other Flagellar Export Apparatus Proteins Govern Type III Secretion Dynamics.

McMurry JL, Minamino T, Furukawa Y, Francis JW, Hill SA, Helms KA, Namba K - PLoS ONE (2015)

FlhBC kinetic survey.Purified T3S proteins were biotinylated and used as ligands. All analyte concentrations were 1 μM. Association and dissociation phases were 180 seconds in all cases. A, FlhBC analyte binding to ligands FlhAC (red), FlhBC (blue), FliH (green), FliI (orange), FliJ (magenta) and FliK (black). B, nonspecific binding control of analyte apparatus proteins binding to sensor-tethered biotinylated BSA. Analyte colors are the same as ligand colors in A. C, Analyte apparatus proteins binding to ligand FlhBC. Colors are the same as in B.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526367&req=5

pone.0134884.g001: FlhBC kinetic survey.Purified T3S proteins were biotinylated and used as ligands. All analyte concentrations were 1 μM. Association and dissociation phases were 180 seconds in all cases. A, FlhBC analyte binding to ligands FlhAC (red), FlhBC (blue), FliH (green), FliI (orange), FliJ (magenta) and FliK (black). B, nonspecific binding control of analyte apparatus proteins binding to sensor-tethered biotinylated BSA. Analyte colors are the same as ligand colors in A. C, Analyte apparatus proteins binding to ligand FlhBC. Colors are the same as in B.
Mentions: 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). A starkly different shift profile from nonspecific control binding (Fig 1B, black trace) was observed for FliK. Smaller differences were noted for all other export proteins, e.g. a small amplitude fast on state for FlhAC, indicating some interaction with FlhBC. Observed binding was complex and nonspecific binding (NSB) as evidenced by response to BSA as ligand was in many cases significant. FliJ, FliH and FlhBC ligands also exhibited binding different from BSA, but were resistant to further analysis due to NSB, low signal and other reasons. Biotinylated FlhBC was tethered to SA sensors and screened for binding versus analyte soluble export apparatus proteins at 1 μM (Fig 1C). Differences in FliI binding were noted. FliK-FlhBC, FlhAC-FlhBC and FlhBC-FliI interactions were selected for further kinetic characterization.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular & Cellular Biology, Kennesaw State University, Kennesaw, Georgia, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus