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Characterization of putative cholesterol recognition/interaction amino acid consensus-like motif of Campylobacter jejuni cytolethal distending toxin C.

Lai CH, Lai CK, Lin YJ, Hung CL, Chu CH, Feng CL, Chang CS, Su HL - PLoS ONE (2013)

Bottom Line: Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain.Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication.These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Medicine, Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan. chl@mail.cmu.edu.tw

ABSTRACT
Cytolethal distending toxin (CDT) produced by Campylobacter jejuni comprises a heterotrimeric complex formed by CdtA, CdtB, and CdtC. Among these toxin subunits, CdtA and CdtC function as essential proteins that mediate toxin binding to cytoplasmic membranes followed by delivery of CdtB into the nucleus. The binding of CdtA/CdtC to the cell surface is mediated by cholesterol, a major component in lipid rafts. Although the putative cholesterol recognition/interaction amino acid consensus (CRAC) domain of CDT has been reported from several bacterial pathogens, the protein regions contributing to CDT binding to cholesterol in C. jejuni remain unclear. Here, we selected a potential CRAC-like region present in the CdtC from C. jejuni for analysis. Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain. Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication. These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.

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Molecular modeling of the interaction of CdtCwt and cholesterol.(A) Schematic representation of a partial sequence of the CRAC-like motif in wild-type and mutant CdtC. The numbers indicate the positions of the amino acid residues. The putative CRAC-like motifs are in boxes. The amino acids in boldface indicate the residues targeted for substitution. (B) Structural model of cholesterol was in complex with putative CRAC of CdtCwt. The cholesterol was shown in stick and colored in blue. The number of amino acids shown in CdtCwt directly interacted with the cholesterol-binding sites. Oxygen atom localized in cholesterol and amino acids were shown in red. (C) Cholesterol showed hydrophobic interactions with L77, P78, F79, G80, Y81, and V82 (boldface lines in green), and formed a hydrogen bond with V82 (dash line in green). Modeling simulation was performed using PyMol, as described in the Materials and Methods.
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pone-0066202-g001: Molecular modeling of the interaction of CdtCwt and cholesterol.(A) Schematic representation of a partial sequence of the CRAC-like motif in wild-type and mutant CdtC. The numbers indicate the positions of the amino acid residues. The putative CRAC-like motifs are in boxes. The amino acids in boldface indicate the residues targeted for substitution. (B) Structural model of cholesterol was in complex with putative CRAC of CdtCwt. The cholesterol was shown in stick and colored in blue. The number of amino acids shown in CdtCwt directly interacted with the cholesterol-binding sites. Oxygen atom localized in cholesterol and amino acids were shown in red. (C) Cholesterol showed hydrophobic interactions with L77, P78, F79, G80, Y81, and V82 (boldface lines in green), and formed a hydrogen bond with V82 (dash line in green). Modeling simulation was performed using PyMol, as described in the Materials and Methods.

Mentions: We recently demonstrated that CDT association with CHO-K1 cells requires intact cholesterol-rich microdomains [16]. A specific conserved sequence, the CRAC motif [L/V(X)1–5Y(X)1–5R/K], may contribute to the association of proteins with cholesterol [22]. To test this, we analyzed the amino acid sequence of CdtC, which contained a putative CRAC-like motif (77LPFGY81VQFTNPK88) (Fig. 1A). To assess whether this CRAC-like motif is required for CdtC binding to lipid rafts and CDT intoxication of cells, we used site-directed mutagenesis to construct a single residue-substituted mutant. The tyrosine residue that plays an important role for protein binding to cholesterol was thus replaced with a proline residue (Y81P). The mutant and wild-type CDT subunits were then subjected to SDS-PAGE (Fig. S1A) and western blot (Fig. S1B) analyses. The purity and protein expression levels of CdtCY81P were similar to those of CdtCwt. The integrity of the toxin complex was then assessed by western blot. As shown in Fig. S1C, both CdtCwt and CdtCY81P can be assembled stably with other holotoxin elements.


Characterization of putative cholesterol recognition/interaction amino acid consensus-like motif of Campylobacter jejuni cytolethal distending toxin C.

Lai CH, Lai CK, Lin YJ, Hung CL, Chu CH, Feng CL, Chang CS, Su HL - PLoS ONE (2013)

Molecular modeling of the interaction of CdtCwt and cholesterol.(A) Schematic representation of a partial sequence of the CRAC-like motif in wild-type and mutant CdtC. The numbers indicate the positions of the amino acid residues. The putative CRAC-like motifs are in boxes. The amino acids in boldface indicate the residues targeted for substitution. (B) Structural model of cholesterol was in complex with putative CRAC of CdtCwt. The cholesterol was shown in stick and colored in blue. The number of amino acids shown in CdtCwt directly interacted with the cholesterol-binding sites. Oxygen atom localized in cholesterol and amino acids were shown in red. (C) Cholesterol showed hydrophobic interactions with L77, P78, F79, G80, Y81, and V82 (boldface lines in green), and formed a hydrogen bond with V82 (dash line in green). Modeling simulation was performed using PyMol, as described in the Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675143&req=5

pone-0066202-g001: Molecular modeling of the interaction of CdtCwt and cholesterol.(A) Schematic representation of a partial sequence of the CRAC-like motif in wild-type and mutant CdtC. The numbers indicate the positions of the amino acid residues. The putative CRAC-like motifs are in boxes. The amino acids in boldface indicate the residues targeted for substitution. (B) Structural model of cholesterol was in complex with putative CRAC of CdtCwt. The cholesterol was shown in stick and colored in blue. The number of amino acids shown in CdtCwt directly interacted with the cholesterol-binding sites. Oxygen atom localized in cholesterol and amino acids were shown in red. (C) Cholesterol showed hydrophobic interactions with L77, P78, F79, G80, Y81, and V82 (boldface lines in green), and formed a hydrogen bond with V82 (dash line in green). Modeling simulation was performed using PyMol, as described in the Materials and Methods.
Mentions: We recently demonstrated that CDT association with CHO-K1 cells requires intact cholesterol-rich microdomains [16]. A specific conserved sequence, the CRAC motif [L/V(X)1–5Y(X)1–5R/K], may contribute to the association of proteins with cholesterol [22]. To test this, we analyzed the amino acid sequence of CdtC, which contained a putative CRAC-like motif (77LPFGY81VQFTNPK88) (Fig. 1A). To assess whether this CRAC-like motif is required for CdtC binding to lipid rafts and CDT intoxication of cells, we used site-directed mutagenesis to construct a single residue-substituted mutant. The tyrosine residue that plays an important role for protein binding to cholesterol was thus replaced with a proline residue (Y81P). The mutant and wild-type CDT subunits were then subjected to SDS-PAGE (Fig. S1A) and western blot (Fig. S1B) analyses. The purity and protein expression levels of CdtCY81P were similar to those of CdtCwt. The integrity of the toxin complex was then assessed by western blot. As shown in Fig. S1C, both CdtCwt and CdtCY81P can be assembled stably with other holotoxin elements.

Bottom Line: Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain.Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication.These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, School of Medicine, Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan. chl@mail.cmu.edu.tw

ABSTRACT
Cytolethal distending toxin (CDT) produced by Campylobacter jejuni comprises a heterotrimeric complex formed by CdtA, CdtB, and CdtC. Among these toxin subunits, CdtA and CdtC function as essential proteins that mediate toxin binding to cytoplasmic membranes followed by delivery of CdtB into the nucleus. The binding of CdtA/CdtC to the cell surface is mediated by cholesterol, a major component in lipid rafts. Although the putative cholesterol recognition/interaction amino acid consensus (CRAC) domain of CDT has been reported from several bacterial pathogens, the protein regions contributing to CDT binding to cholesterol in C. jejuni remain unclear. Here, we selected a potential CRAC-like region present in the CdtC from C. jejuni for analysis. Molecular modeling showed that the predicted functional domain had the shape of a hydrophobic groove, facilitating cholesterol localization to this domain. Mutation of a tyrosine residue in the CRAC-like region decreased direct binding of CdtC to cholesterol rather than toxin intermolecular interactions and led to impaired CDT intoxication. These results provide a molecular link between C. jejuni CdtC and membrane-lipid rafts through the CRAC-like region, which contributes to toxin recognition and interaction with cholesterol.

Show MeSH
Related in: MedlinePlus