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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

FlhAC-FlhBC binding.Ligand FlhAC was exposed to 2, 1, 0.5, 0.25 and 0.125 μM FlhBC. A, association with fits to a one-state model B, dissociation with fits to a global two-state model C, steady state analysis. D, kobs vs. [FlhBC] to estimate kinetic constants, R2 = 0.98.
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pone.0134884.g003: FlhAC-FlhBC binding.Ligand FlhAC was exposed to 2, 1, 0.5, 0.25 and 0.125 μM FlhBC. A, association with fits to a one-state model B, dissociation with fits to a global two-state model C, steady state analysis. D, kobs vs. [FlhBC] to estimate kinetic constants, R2 = 0.98.

Mentions: Ligand FlhAC-analyte FlhBC binding also exhibited complexity and did not fit global one-state association-then-dissociation models. Single exponentials did fit the association phase (Fig 3A). Global two-state exponentials could fit dissociation with koffs of 0.13 s−1 and 4.7 x 10−3 s−1 (Fig 3B). Saturation analysis (Fig 3C) yielded a KD of 1.1 μM. Plotting kobs vs [FlhBC](Fig 3D) led to an estimate of kon of 8.5 x 104 M−1s−1 and thus a nominal one-state koff of 0.09 s−1, though caution should accompany interpretation of these values (see Discussion).


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)

FlhAC-FlhBC binding.Ligand FlhAC was exposed to 2, 1, 0.5, 0.25 and 0.125 μM FlhBC. A, association with fits to a one-state model B, dissociation with fits to a global two-state model C, steady state analysis. D, kobs vs. [FlhBC] to estimate kinetic constants, R2 = 0.98.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134884.g003: FlhAC-FlhBC binding.Ligand FlhAC was exposed to 2, 1, 0.5, 0.25 and 0.125 μM FlhBC. A, association with fits to a one-state model B, dissociation with fits to a global two-state model C, steady state analysis. D, kobs vs. [FlhBC] to estimate kinetic constants, R2 = 0.98.
Mentions: Ligand FlhAC-analyte FlhBC binding also exhibited complexity and did not fit global one-state association-then-dissociation models. Single exponentials did fit the association phase (Fig 3A). Global two-state exponentials could fit dissociation with koffs of 0.13 s−1 and 4.7 x 10−3 s−1 (Fig 3B). Saturation analysis (Fig 3C) yielded a KD of 1.1 μM. Plotting kobs vs [FlhBC](Fig 3D) led to an estimate of kon of 8.5 x 104 M−1s−1 and thus a nominal one-state koff of 0.09 s−1, though caution should accompany interpretation of these values (see Discussion).

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