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 coil strains, deamidates t he RhoA GTP-binding protein glutamine 63 and impairs RhoGAP-mediated GTP hy drolysis resulting in RhoA permanent activation. Using peptides derived fro m the RhoA sequence, we found that DTAGQEDYDRL (corresponding to RhoA 59-69 residues) was the minimum RhoA-derived peptide which could be deamidated i n vitro by the CNF1 catalytic domain (CNF1-Cter), Site-directed mutagenesis outside the RhoA 59-69 sequence had no influence on glutamine 63 deamidati on 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 DYD RL 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. (C) 2000 Academic Press.