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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

Full-length FlhB forms a dimer.A, anti-FlhB immunoblot of hook-basal body preparation (HBB) and purified FlhB(N269A). Approximate locations of molecular weight standards in kDa are shown at left. B, sedimentation equilibrium analytical centrifugation. A fit is shown to a single-species model, the molecular weight of which is 84.1 kDa (monomer of tagged FlhB(N269A) = 42.3 kDa).
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pone.0134884.g005: Full-length FlhB forms a dimer.A, anti-FlhB immunoblot of hook-basal body preparation (HBB) and purified FlhB(N269A). Approximate locations of molecular weight standards in kDa are shown at left. B, sedimentation equilibrium analytical centrifugation. A fit is shown to a single-species model, the molecular weight of which is 84.1 kDa (monomer of tagged FlhB(N269A) = 42.3 kDa).

Mentions: FlhBC-FlhBC interactions (Fig 1) were at best minimally observable, consistent with earlier studies that found questionable or no interaction [12,21]. We report here purification of solubilized FlhB under non-denaturing conditions using a procedure modified from a prior method used to purify FlhA [22]. The uncleavable but export competent N269A variant [14] was used to assure retention of the carboxyl-terminal subdomain in the solubilizing conditions used (though later purification of wild-type FlhB from pMM9, which complements a flhB , showed that the subdomain consisting of residues 270–383 is retained (S1 Fig)). Anti-FlhB immunoblots of hook-basal bodies (HBBs) prepared from SJW880 [28] under conditions in which the C ring and export apparatus proteins are retained [29] (gift from Noreen R. Francis) demonstrated significant SDS-stable dimerization, as did purified FlhB(N269A) (Fig 5A). Full-length FlhB(N269A), solubilized in the neutrally buoyant, nondenaturing detergent C8E5, formed a stable dimer in a sedimentation equilibrium ultracenrifugation experiment (Fig 5B). Fits to a single species model produced a molecular weight of 84.1 kDa, consistent with a FlhB dimer. The tagged monomer is ~42.3 kDa.


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)

Full-length FlhB forms a dimer.A, anti-FlhB immunoblot of hook-basal body preparation (HBB) and purified FlhB(N269A). Approximate locations of molecular weight standards in kDa are shown at left. B, sedimentation equilibrium analytical centrifugation. A fit is shown to a single-species model, the molecular weight of which is 84.1 kDa (monomer of tagged FlhB(N269A) = 42.3 kDa).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134884.g005: Full-length FlhB forms a dimer.A, anti-FlhB immunoblot of hook-basal body preparation (HBB) and purified FlhB(N269A). Approximate locations of molecular weight standards in kDa are shown at left. B, sedimentation equilibrium analytical centrifugation. A fit is shown to a single-species model, the molecular weight of which is 84.1 kDa (monomer of tagged FlhB(N269A) = 42.3 kDa).
Mentions: FlhBC-FlhBC interactions (Fig 1) were at best minimally observable, consistent with earlier studies that found questionable or no interaction [12,21]. We report here purification of solubilized FlhB under non-denaturing conditions using a procedure modified from a prior method used to purify FlhA [22]. The uncleavable but export competent N269A variant [14] was used to assure retention of the carboxyl-terminal subdomain in the solubilizing conditions used (though later purification of wild-type FlhB from pMM9, which complements a flhB , showed that the subdomain consisting of residues 270–383 is retained (S1 Fig)). Anti-FlhB immunoblots of hook-basal bodies (HBBs) prepared from SJW880 [28] under conditions in which the C ring and export apparatus proteins are retained [29] (gift from Noreen R. Francis) demonstrated significant SDS-stable dimerization, as did purified FlhB(N269A) (Fig 5A). Full-length FlhB(N269A), solubilized in the neutrally buoyant, nondenaturing detergent C8E5, formed a stable dimer in a sedimentation equilibrium ultracenrifugation experiment (Fig 5B). Fits to a single species model produced a molecular weight of 84.1 kDa, consistent with a FlhB dimer. The tagged monomer is ~42.3 kDa.

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