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A gatekeeper chaperone complex directs translocator secretion during type three secretion.

Archuleta TL, Spiller BW - PLoS Pathog. (2014)

Bottom Line: A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective.One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion.The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown.

View Article: PubMed Central - PubMed

Affiliation: Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

ABSTRACT
Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

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The Scc3-CopNΔ84 complex binds directly to translocators.A. An overlay of class II T3S chaperones. All structures except the Scc3 structure were determined bound to translocator peptides. For clarity, only the IpaB peptide from Shigella is shown. Scc3 is shown in salmon, IpgC (Shigella) in teal, PcrH (Pseudomonas) in green, and SycD (Yersinia) in blue. The peptide-binding site is conserved and open in Scc3. B. CopN-Scc3 complex directly binds to the CopB translocator. Top: gel filtration traces reveal that the ternary complex is tight enough to survive gel filtration. Bottom: SDS PAGE confirming complex formation.
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ppat-1004498-g003: The Scc3-CopNΔ84 complex binds directly to translocators.A. An overlay of class II T3S chaperones. All structures except the Scc3 structure were determined bound to translocator peptides. For clarity, only the IpaB peptide from Shigella is shown. Scc3 is shown in salmon, IpgC (Shigella) in teal, PcrH (Pseudomonas) in green, and SycD (Yersinia) in blue. The peptide-binding site is conserved and open in Scc3. B. CopN-Scc3 complex directly binds to the CopB translocator. Top: gel filtration traces reveal that the ternary complex is tight enough to survive gel filtration. Bottom: SDS PAGE confirming complex formation.

Mentions: TPR family proteins are often unfolded when not bound to appropriate ligands and are considered to be somewhat flexible proteins [40], [41]. Scc3 has an appropriately organized but empty binding cleft when bound to CopN. Structural comparison with other class II chaperones, for which structures have been determined in complex with translocator-derived peptides, indicates that CopNΔ84 binding causes no significant reorganization of the translocator-binding site (Figure 3A). The Scc3-CopNΔ84 binding mode leaves the translocator-binding site on Scc3 unperturbed and available to bind translocators (Figure 3A). In support of this observation, the purified Scc3-CopNΔ84 complex is able to directly bind a translocator-derived peptide (presented as residues 158–177 from CopB fused to GST) and form a CopNΔ84-Scc3-CopB158-177 complex as judged by size exclusion chromatography (Figure 3B). Isothermal Titration Calorimetry (ITC) using a synthetic peptide (CopB residues 163–173) revealed Kd's of 79±16 µM for the Scc3-CopB peptide complex and 49±13 µM for the Scc3-CopNΔ84 peptide complex (Supporting Figure S5, Methods S1).


A gatekeeper chaperone complex directs translocator secretion during type three secretion.

Archuleta TL, Spiller BW - PLoS Pathog. (2014)

The Scc3-CopNΔ84 complex binds directly to translocators.A. An overlay of class II T3S chaperones. All structures except the Scc3 structure were determined bound to translocator peptides. For clarity, only the IpaB peptide from Shigella is shown. Scc3 is shown in salmon, IpgC (Shigella) in teal, PcrH (Pseudomonas) in green, and SycD (Yersinia) in blue. The peptide-binding site is conserved and open in Scc3. B. CopN-Scc3 complex directly binds to the CopB translocator. Top: gel filtration traces reveal that the ternary complex is tight enough to survive gel filtration. Bottom: SDS PAGE confirming complex formation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222845&req=5

ppat-1004498-g003: The Scc3-CopNΔ84 complex binds directly to translocators.A. An overlay of class II T3S chaperones. All structures except the Scc3 structure were determined bound to translocator peptides. For clarity, only the IpaB peptide from Shigella is shown. Scc3 is shown in salmon, IpgC (Shigella) in teal, PcrH (Pseudomonas) in green, and SycD (Yersinia) in blue. The peptide-binding site is conserved and open in Scc3. B. CopN-Scc3 complex directly binds to the CopB translocator. Top: gel filtration traces reveal that the ternary complex is tight enough to survive gel filtration. Bottom: SDS PAGE confirming complex formation.
Mentions: TPR family proteins are often unfolded when not bound to appropriate ligands and are considered to be somewhat flexible proteins [40], [41]. Scc3 has an appropriately organized but empty binding cleft when bound to CopN. Structural comparison with other class II chaperones, for which structures have been determined in complex with translocator-derived peptides, indicates that CopNΔ84 binding causes no significant reorganization of the translocator-binding site (Figure 3A). The Scc3-CopNΔ84 binding mode leaves the translocator-binding site on Scc3 unperturbed and available to bind translocators (Figure 3A). In support of this observation, the purified Scc3-CopNΔ84 complex is able to directly bind a translocator-derived peptide (presented as residues 158–177 from CopB fused to GST) and form a CopNΔ84-Scc3-CopB158-177 complex as judged by size exclusion chromatography (Figure 3B). Isothermal Titration Calorimetry (ITC) using a synthetic peptide (CopB residues 163–173) revealed Kd's of 79±16 µM for the Scc3-CopB peptide complex and 49±13 µM for the Scc3-CopNΔ84 peptide complex (Supporting Figure S5, Methods S1).

Bottom Line: A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective.One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion.The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown.

View Article: PubMed Central - PubMed

Affiliation: Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

ABSTRACT
Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

Show MeSH
Related in: MedlinePlus