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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

Steady state analysis of FliH binding to full length wild-type FlhB.Association phases from which steady state amplitudes were determined are shown in the inset. FliH concentrations ranged from 0.125 to 8 μM.
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pone.0134884.g006: Steady state analysis of FliH binding to full length wild-type FlhB.Association phases from which steady state amplitudes were determined are shown in the inset. FliH concentrations ranged from 0.125 to 8 μM.

Mentions: Purified wild-type FlhB exhibited specific binding to FliH as ligand. Dissociation anomalies perhaps due to detergent effects prevented kinetic analysis. KD determined from steady state analysis (Fig 6) was 0.8 μM. The 2 μM sample was excluded from analysis due to anomalous readings from that channel, though its inclusion would render a KD of 0.9 μM with a concomitant reduction in R2 from 0.99 to 0.82. Further experiments with other analytes were precluded by instability of the FlhB preparations; we hope to examine them in future studies.


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)

Steady state analysis of FliH binding to full length wild-type FlhB.Association phases from which steady state amplitudes were determined are shown in the inset. FliH concentrations ranged from 0.125 to 8 μM.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134884.g006: Steady state analysis of FliH binding to full length wild-type FlhB.Association phases from which steady state amplitudes were determined are shown in the inset. FliH concentrations ranged from 0.125 to 8 μM.
Mentions: Purified wild-type FlhB exhibited specific binding to FliH as ligand. Dissociation anomalies perhaps due to detergent effects prevented kinetic analysis. KD determined from steady state analysis (Fig 6) was 0.8 μM. The 2 μM sample was excluded from analysis due to anomalous readings from that channel, though its inclusion would render a KD of 0.9 μM with a concomitant reduction in R2 from 0.99 to 0.82. Further experiments with other analytes were precluded by instability of the FlhB preparations; we hope to examine them in future studies.

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