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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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Crystal structure of Scc3-CopNΔ84.CopN is colored green, with the YopN homology region in dark green and the TyeA homology region in light green. A. A ribbon diagram of the Scc3-CopNΔ84 structure. Approximate domain boundaries are indicated. Scc3, salmon, binds across the domain 2-domain 3 domain interface. B. A close-up of the Scc3-CopNΔ84 interface, oriented as in A, with Scc3 shown as a molecular surface. Scc3 forms a relatively flat surface that bridges domains 2 and 3 of CopNΔ84.C., D. Comparisons of CopN and homologs. C. Comparison between MxiC and CopN. MxiC is colored tan and shown with thin helices. D. Comparison between CopN and the YopN/TyeA complex. YopN is tan and TyeA is brown. YopN and TyeA are and shown with thin helices. E. Overlay of TyeA in orientation shown in D. and when aligned as a rigid body to the carboxy terminal 91 residues of CopN (rmsd = 0.4 Å).
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ppat-1004498-g001: Crystal structure of Scc3-CopNΔ84.CopN is colored green, with the YopN homology region in dark green and the TyeA homology region in light green. A. A ribbon diagram of the Scc3-CopNΔ84 structure. Approximate domain boundaries are indicated. Scc3, salmon, binds across the domain 2-domain 3 domain interface. B. A close-up of the Scc3-CopNΔ84 interface, oriented as in A, with Scc3 shown as a molecular surface. Scc3 forms a relatively flat surface that bridges domains 2 and 3 of CopNΔ84.C., D. Comparisons of CopN and homologs. C. Comparison between MxiC and CopN. MxiC is colored tan and shown with thin helices. D. Comparison between CopN and the YopN/TyeA complex. YopN is tan and TyeA is brown. YopN and TyeA are and shown with thin helices. E. Overlay of TyeA in orientation shown in D. and when aligned as a rigid body to the carboxy terminal 91 residues of CopN (rmsd = 0.4 Å).

Mentions: We determined the crystal structure of the Scc3-CopNΔ84 complex and refined the structure to 2.2 Å (PDB ID 4NRH). The amino terminal 84 residues of CopN were not included in the construct used for crystallization because they are unstructured [25]. Data collection and structure refinement statistics are given in Table 1, and representative electron density is shown in Supporting Figure S1. Two nearly identical Scc3-CopNΔ84 complexes (RMS deviation 0.34 Å for all CopNΔ84 mainchain atoms and 0.49 Å for all Scc3 mainchain atoms) are present in the asymmetric unit. CopNΔ84 forms a long cylindrical structure composed of three helical domains (Figure 1). A search for structurally similar proteins using the DALI software [33], indicates structural homology to the globin fold, which, aside from the use described here, is used in bacteria both as a light harvesting complex and as a stress response sigma factor [34]–[36]. In gatekeeper proteins multiple domains are concatenated through elongated connecting helices, whereas globin domains typically oligomerize through lateral contacts. CopNΔ84 is structurally similar to other gatekeeper proteins, both MxiC from Shigella and the YopN-TyeA complex from Yersinia. The most substantial differences among family members relate to the position of the carboxy-terminal domain or subunit (Figure 1 and [37], [38]). In the Scc3-CopNΔ84 complex, this domain is translated ∼9.5 Å and rotated ∼50° relative to the YopN-TyeA complex (Figure 1). Similarly, Scc3 is structurally similar to other translocator chaperones. The striking result from the Scc3-CopNΔ84 is the unexpected assembly of the complex and the role of the Scc3 amino terminus in binding CopNΔ84.


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

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

Crystal structure of Scc3-CopNΔ84.CopN is colored green, with the YopN homology region in dark green and the TyeA homology region in light green. A. A ribbon diagram of the Scc3-CopNΔ84 structure. Approximate domain boundaries are indicated. Scc3, salmon, binds across the domain 2-domain 3 domain interface. B. A close-up of the Scc3-CopNΔ84 interface, oriented as in A, with Scc3 shown as a molecular surface. Scc3 forms a relatively flat surface that bridges domains 2 and 3 of CopNΔ84.C., D. Comparisons of CopN and homologs. C. Comparison between MxiC and CopN. MxiC is colored tan and shown with thin helices. D. Comparison between CopN and the YopN/TyeA complex. YopN is tan and TyeA is brown. YopN and TyeA are and shown with thin helices. E. Overlay of TyeA in orientation shown in D. and when aligned as a rigid body to the carboxy terminal 91 residues of CopN (rmsd = 0.4 Å).
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Related In: Results  -  Collection

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ppat-1004498-g001: Crystal structure of Scc3-CopNΔ84.CopN is colored green, with the YopN homology region in dark green and the TyeA homology region in light green. A. A ribbon diagram of the Scc3-CopNΔ84 structure. Approximate domain boundaries are indicated. Scc3, salmon, binds across the domain 2-domain 3 domain interface. B. A close-up of the Scc3-CopNΔ84 interface, oriented as in A, with Scc3 shown as a molecular surface. Scc3 forms a relatively flat surface that bridges domains 2 and 3 of CopNΔ84.C., D. Comparisons of CopN and homologs. C. Comparison between MxiC and CopN. MxiC is colored tan and shown with thin helices. D. Comparison between CopN and the YopN/TyeA complex. YopN is tan and TyeA is brown. YopN and TyeA are and shown with thin helices. E. Overlay of TyeA in orientation shown in D. and when aligned as a rigid body to the carboxy terminal 91 residues of CopN (rmsd = 0.4 Å).
Mentions: We determined the crystal structure of the Scc3-CopNΔ84 complex and refined the structure to 2.2 Å (PDB ID 4NRH). The amino terminal 84 residues of CopN were not included in the construct used for crystallization because they are unstructured [25]. Data collection and structure refinement statistics are given in Table 1, and representative electron density is shown in Supporting Figure S1. Two nearly identical Scc3-CopNΔ84 complexes (RMS deviation 0.34 Å for all CopNΔ84 mainchain atoms and 0.49 Å for all Scc3 mainchain atoms) are present in the asymmetric unit. CopNΔ84 forms a long cylindrical structure composed of three helical domains (Figure 1). A search for structurally similar proteins using the DALI software [33], indicates structural homology to the globin fold, which, aside from the use described here, is used in bacteria both as a light harvesting complex and as a stress response sigma factor [34]–[36]. In gatekeeper proteins multiple domains are concatenated through elongated connecting helices, whereas globin domains typically oligomerize through lateral contacts. CopNΔ84 is structurally similar to other gatekeeper proteins, both MxiC from Shigella and the YopN-TyeA complex from Yersinia. The most substantial differences among family members relate to the position of the carboxy-terminal domain or subunit (Figure 1 and [37], [38]). In the Scc3-CopNΔ84 complex, this domain is translated ∼9.5 Å and rotated ∼50° relative to the YopN-TyeA complex (Figure 1). Similarly, Scc3 is structurally similar to other translocator chaperones. The striking result from the Scc3-CopNΔ84 is the unexpected assembly of the complex and the role of the Scc3 amino terminus in binding CopNΔ84.

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