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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

Simulation of FliK-FlhBC binding.A and B, association and dissociation phases. Concentrations of FlhBC were 5, 4, 3, 2, 1, 0.5, 0.25, and 0 μM. The 0.5 and 0.25 μM traces are unlabeled. Fits to individual two-state exponentials are shown as red lines. C-G, Simulations of the 5, 4, 3, 2 and 1 μM data with a conformational change model using global parameters for koff and kon and koff for transition to the conformationally altered state (Table 2). H, Apparent kon vs [FlhBC] to determine the global kon (= slope).
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pone.0134884.g002: Simulation of FliK-FlhBC binding.A and B, association and dissociation phases. Concentrations of FlhBC were 5, 4, 3, 2, 1, 0.5, 0.25, and 0 μM. The 0.5 and 0.25 μM traces are unlabeled. Fits to individual two-state exponentials are shown as red lines. C-G, Simulations of the 5, 4, 3, 2 and 1 μM data with a conformational change model using global parameters for koff and kon and koff for transition to the conformationally altered state (Table 2). H, Apparent kon vs [FlhBC] to determine the global kon (= slope).

Mentions: We previously reported a KD of 3.2 μM for FliK-FlhBC binding determined by steady state analysis of SPR data [13]. Delving further into the complexity to better understand the kinetics, BLI sensorgrams were collected for a concentration course ranging from 0 to 5 μM FlhBC. As shown in Fig 2A and 2B, association and dissociation phases could be fit by two exponentials, i.e. parallel events, but there was no global solution that yielded constants that fit two independent states. Instead, numerical simulations were performed using differential equations constructed from a conformational change model. Simulations of association-then-dissociation are shown for 5, 4, 3, 2 and 1 μM in Fig 2C–2G. Parameters for constants used in the simulations are shown in Table 1 and include slow transitions to and from the AB* state. Plotting k1 determined from simulations, which is equivalent to the observed rate constant (kobs) for initial binding in that it also accounts for dissociation occurring during the association phase, vs. analyte concentration (Fig 2H) yielded kon of 5.5 x 104 M−1s−1. Combination with a koff of 0.44 s−1 gave a KD of 8.0 μM for the initial binding event, consistent with our earlier study. Supporting the conformational change model is the observation that the amplitude of the slow-off state in the dissociation phase varied proportionately with the length of the association phase (S2 Fig). It should be noted that amplitude variations between full kinetic characterizations and the Fig 1 survey are likely a function of different specific binding activities of different preparations for both ligand and analyte. All concentration courses in this experiment were done with dilutions of the same preparation. We also note that the overall KD determined by steady state analysis, i.e. including the slow states, for FliK-FlhBC in Fig 2 is 2.1 μM S3 Fig), almost identical to that of the preparations used in the earlier report despite very different amplitudes.


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)

Simulation of FliK-FlhBC binding.A and B, association and dissociation phases. Concentrations of FlhBC were 5, 4, 3, 2, 1, 0.5, 0.25, and 0 μM. The 0.5 and 0.25 μM traces are unlabeled. Fits to individual two-state exponentials are shown as red lines. C-G, Simulations of the 5, 4, 3, 2 and 1 μM data with a conformational change model using global parameters for koff and kon and koff for transition to the conformationally altered state (Table 2). H, Apparent kon vs [FlhBC] to determine the global kon (= slope).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134884.g002: Simulation of FliK-FlhBC binding.A and B, association and dissociation phases. Concentrations of FlhBC were 5, 4, 3, 2, 1, 0.5, 0.25, and 0 μM. The 0.5 and 0.25 μM traces are unlabeled. Fits to individual two-state exponentials are shown as red lines. C-G, Simulations of the 5, 4, 3, 2 and 1 μM data with a conformational change model using global parameters for koff and kon and koff for transition to the conformationally altered state (Table 2). H, Apparent kon vs [FlhBC] to determine the global kon (= slope).
Mentions: We previously reported a KD of 3.2 μM for FliK-FlhBC binding determined by steady state analysis of SPR data [13]. Delving further into the complexity to better understand the kinetics, BLI sensorgrams were collected for a concentration course ranging from 0 to 5 μM FlhBC. As shown in Fig 2A and 2B, association and dissociation phases could be fit by two exponentials, i.e. parallel events, but there was no global solution that yielded constants that fit two independent states. Instead, numerical simulations were performed using differential equations constructed from a conformational change model. Simulations of association-then-dissociation are shown for 5, 4, 3, 2 and 1 μM in Fig 2C–2G. Parameters for constants used in the simulations are shown in Table 1 and include slow transitions to and from the AB* state. Plotting k1 determined from simulations, which is equivalent to the observed rate constant (kobs) for initial binding in that it also accounts for dissociation occurring during the association phase, vs. analyte concentration (Fig 2H) yielded kon of 5.5 x 104 M−1s−1. Combination with a koff of 0.44 s−1 gave a KD of 8.0 μM for the initial binding event, consistent with our earlier study. Supporting the conformational change model is the observation that the amplitude of the slow-off state in the dissociation phase varied proportionately with the length of the association phase (S2 Fig). It should be noted that amplitude variations between full kinetic characterizations and the Fig 1 survey are likely a function of different specific binding activities of different preparations for both ligand and analyte. All concentration courses in this experiment were done with dilutions of the same preparation. We also note that the overall KD determined by steady state analysis, i.e. including the slow states, for FliK-FlhBC in Fig 2 is 2.1 μM S3 Fig), almost identical to that of the preparations used in the earlier report despite very different amplitudes.

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