Deamidation of RhoA glutamine 63 by the Escherichia coli CNF1 toxin requires a short sequence of the GTPase switch 2 domain.
CNF1, a toxin produced by pathogenic Escherichia coli strains, deamidates the RhoA GTP-binding protein glutamine 63 and impairs RhoGAP-mediated GTP hydrolysis resulting in RhoA permanent activation. Using peptides derived from the RhoA sequence, we found that DTAGQEDYDRL (corresponding to RhoA 59-69 residues) was the minimum RhoA-derived peptide which could be deamidated in vitro by the CNF1 catalytic domain (CNF1-Cter). Site-directed mutagenesis outside the RhoA 59-69 sequence had no influence on glutamine 63 deamidation by CNF1-Cter. RhoA proteins with substitutions L57G, D65G, Y66G, or R70G were not affected in their ability to be deamidated by CNF1-Cter, whereas this was abolished by the R68G substitution. Arginine 68 is part of the DYDRL motif that is strictly conserved in Rho, Rac, and Cdc42 but not in other small GTP-binding proteins consistent with the observation that only Rho, Rac, and Cdc42 can be modified by CNF1.
Flatau
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INSERM U 452, Faculté de Médecine, 28 Avenue de Valombrose, Nice Cedex 2, 06107, France.
Landraud
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Boquet
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Bruzzone
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Munro
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D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D003603:Cytotoxins / Q000378:metabolism / Q000633:toxicity*; D004926:Escherichia coli / Q000378:metabolism / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D005973:Glutamine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D005973:Glutamine; C060296:cytotoxic necrotizing factor type 1; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein