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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-FliI binding.FlhBC was used as ligand to examine binding of FliI in the absence and presence of ATP. A, Binding of 4, 2, 1, 0.5 and 0.25 μM FliI with fits to a global one-state association-then-dissociation model shown in red. The green points are 4μM FliI exposed to a sensor without FlhBC, e.g. NSB. B, FlhBC-FliI binding with FliI preequilibrated with 5 mM ATP and 5 mM MgCl2 added to all phases. FliI concentrations were 2, 1, 0.5, 0.25, 0.13, 0.062 and 0 μM. Green points represent the 2 uM sample binding to sensor without ligand. C, Steady state analydis of FliI with ATP from panel B, KD = 1.1 μM.
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pone.0134884.g004: FlhBC-FliI binding.FlhBC was used as ligand to examine binding of FliI in the absence and presence of ATP. A, Binding of 4, 2, 1, 0.5 and 0.25 μM FliI with fits to a global one-state association-then-dissociation model shown in red. The green points are 4μM FliI exposed to a sensor without FlhBC, e.g. NSB. B, FlhBC-FliI binding with FliI preequilibrated with 5 mM ATP and 5 mM MgCl2 added to all phases. FliI concentrations were 2, 1, 0.5, 0.25, 0.13, 0.062 and 0 μM. Green points represent the 2 uM sample binding to sensor without ligand. C, Steady state analydis of FliI with ATP from panel B, KD = 1.1 μM.

Mentions: FlhB binds FliI via its cytoplasmic domain. As shown in Fig 4, ligand FlhBC bound FliI both in the absence and presence of Mg2+-ATP. For FliI without ATP (Fig 4A), fits to a global one-state model yielded a KD of 84 nM with a kon of 1.8 x 104 M−1s−1 and koff of 1.5 x 10−3 s−1. Preincubation of analyte FliI with an excess of ATP resulted in near elimination of nonspecific binding and more complex kinetics; substantial fast-on and fast off-states are seen relative to the no ATP sample. Data do not fit two-state models, perhaps reflecting additional states induced by oligomerization of FliI (see Discussion). KD determined from steady state analysis (Fig 4C) is 1.1 μM.


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-FliI binding.FlhBC was used as ligand to examine binding of FliI in the absence and presence of ATP. A, Binding of 4, 2, 1, 0.5 and 0.25 μM FliI with fits to a global one-state association-then-dissociation model shown in red. The green points are 4μM FliI exposed to a sensor without FlhBC, e.g. NSB. B, FlhBC-FliI binding with FliI preequilibrated with 5 mM ATP and 5 mM MgCl2 added to all phases. FliI concentrations were 2, 1, 0.5, 0.25, 0.13, 0.062 and 0 μM. Green points represent the 2 uM sample binding to sensor without ligand. C, Steady state analydis of FliI with ATP from panel B, KD = 1.1 μM.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134884.g004: FlhBC-FliI binding.FlhBC was used as ligand to examine binding of FliI in the absence and presence of ATP. A, Binding of 4, 2, 1, 0.5 and 0.25 μM FliI with fits to a global one-state association-then-dissociation model shown in red. The green points are 4μM FliI exposed to a sensor without FlhBC, e.g. NSB. B, FlhBC-FliI binding with FliI preequilibrated with 5 mM ATP and 5 mM MgCl2 added to all phases. FliI concentrations were 2, 1, 0.5, 0.25, 0.13, 0.062 and 0 μM. Green points represent the 2 uM sample binding to sensor without ligand. C, Steady state analydis of FliI with ATP from panel B, KD = 1.1 μM.
Mentions: FlhB binds FliI via its cytoplasmic domain. As shown in Fig 4, ligand FlhBC bound FliI both in the absence and presence of Mg2+-ATP. For FliI without ATP (Fig 4A), fits to a global one-state model yielded a KD of 84 nM with a kon of 1.8 x 104 M−1s−1 and koff of 1.5 x 10−3 s−1. Preincubation of analyte FliI with an excess of ATP resulted in near elimination of nonspecific binding and more complex kinetics; substantial fast-on and fast off-states are seen relative to the no ATP sample. Data do not fit two-state models, perhaps reflecting additional states induced by oligomerization of FliI (see Discussion). KD determined from steady state analysis (Fig 4C) is 1.1 μM.

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