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The Shigella type three secretion system effector OspG directly and specifically binds to host ubiquitin for activation.

Zhou Y, Dong N, Hu L, Shao F - PLoS ONE (2013)

Bottom Line: OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase.GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding.Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, Beijing, China.

ABSTRACT
The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.

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Secondary structure-based sequence alignment of OspG with human JNK3.The protein names are indicated at the left of the alignment. The key residues in the kinase domain are colored in red. Predicted secondary structures of OspG are shown on top of the OspG sequence. Secondary structures determined from crystal structure of JNK3 are shown underneath the sequence of JNK3 in the alignment. Green ovals are α helices and blue arrows are β strands. The sub-domains are labeled with Roman numerals.
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pone-0057558-g004: Secondary structure-based sequence alignment of OspG with human JNK3.The protein names are indicated at the left of the alignment. The key residues in the kinase domain are colored in red. Predicted secondary structures of OspG are shown on top of the OspG sequence. Secondary structures determined from crystal structure of JNK3 are shown underneath the sequence of JNK3 in the alignment. Green ovals are α helices and blue arrows are β strands. The sub-domains are labeled with Roman numerals.

Mentions: OspG is a small-size protein with only 196 amino acids and its sequence from residue 24 to 160 exhibits confident similarities to sub-domains I to VII of eukaryotic serine/threonine kinase (Fig. 3A and Fig. 4). The extreme carboxyl terminus (residues 161–196), predicted to form an α helix, does not show any sequence similarity to known proteins. The fact that no classical ubiquitin-binding domains or motifs could be identified in OspG indicates a novel ubiquitin-binding mode and also promoted us to further investigate its ubiquitin binding mechanism. Truncation of the amino terminal 21 residues preceding the putative kinase domain did not affect OspG binding to ubiquitin in the in vitro pulldown assay (Fig. 3B). Notably, removal of the extreme carboxyl terminal 26 residues in OspG completely abolished its interaction with ubiquitin (Fig. 3C), suggesting that the carboxyl terminal region following the kinase-like domain in OspG is essential for ubiquitin binding. Mutation of two leucine residues (L190D/L191D) in this region abolishedOspG binding to ubiquitin (Fig. 3C), suggesting the importance of these two residues. Furthermore, when the carboxyl terminal 26, 31, or 35 residues were artificially fused to GST protein and the fusion proteins were assayed for co-precipitation by ubiquitin, none of the three chimeric proteins were capable of binding to ubiquitin (Fig. 3D). These data suggest that the carboxyl terminal helical region in OspG is essential but not sufficient for mediating specific interaction with ubiquitin and the kinase-like domain is also involved in ubiquitin binding.


The Shigella type three secretion system effector OspG directly and specifically binds to host ubiquitin for activation.

Zhou Y, Dong N, Hu L, Shao F - PLoS ONE (2013)

Secondary structure-based sequence alignment of OspG with human JNK3.The protein names are indicated at the left of the alignment. The key residues in the kinase domain are colored in red. Predicted secondary structures of OspG are shown on top of the OspG sequence. Secondary structures determined from crystal structure of JNK3 are shown underneath the sequence of JNK3 in the alignment. Green ovals are α helices and blue arrows are β strands. The sub-domains are labeled with Roman numerals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585378&req=5

pone-0057558-g004: Secondary structure-based sequence alignment of OspG with human JNK3.The protein names are indicated at the left of the alignment. The key residues in the kinase domain are colored in red. Predicted secondary structures of OspG are shown on top of the OspG sequence. Secondary structures determined from crystal structure of JNK3 are shown underneath the sequence of JNK3 in the alignment. Green ovals are α helices and blue arrows are β strands. The sub-domains are labeled with Roman numerals.
Mentions: OspG is a small-size protein with only 196 amino acids and its sequence from residue 24 to 160 exhibits confident similarities to sub-domains I to VII of eukaryotic serine/threonine kinase (Fig. 3A and Fig. 4). The extreme carboxyl terminus (residues 161–196), predicted to form an α helix, does not show any sequence similarity to known proteins. The fact that no classical ubiquitin-binding domains or motifs could be identified in OspG indicates a novel ubiquitin-binding mode and also promoted us to further investigate its ubiquitin binding mechanism. Truncation of the amino terminal 21 residues preceding the putative kinase domain did not affect OspG binding to ubiquitin in the in vitro pulldown assay (Fig. 3B). Notably, removal of the extreme carboxyl terminal 26 residues in OspG completely abolished its interaction with ubiquitin (Fig. 3C), suggesting that the carboxyl terminal region following the kinase-like domain in OspG is essential for ubiquitin binding. Mutation of two leucine residues (L190D/L191D) in this region abolishedOspG binding to ubiquitin (Fig. 3C), suggesting the importance of these two residues. Furthermore, when the carboxyl terminal 26, 31, or 35 residues were artificially fused to GST protein and the fusion proteins were assayed for co-precipitation by ubiquitin, none of the three chimeric proteins were capable of binding to ubiquitin (Fig. 3D). These data suggest that the carboxyl terminal helical region in OspG is essential but not sufficient for mediating specific interaction with ubiquitin and the kinase-like domain is also involved in ubiquitin binding.

Bottom Line: OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase.GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding.Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, Beijing, China.

ABSTRACT
The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.

Show MeSH
Related in: MedlinePlus