Effect of Mutational Alteration of Asn-128 in the Putative GTP-Binding Domain of Tetracycline Resistance Determinant

The deduced amino acid sequence of Campylobacterjejuni Tet(O), cloned in Escherichia coli, has shown that it contains the five highly conserved sequences of the GTP-binding domain found in other GTPases. Asn-128 belongs to the G4 motif of such a domain and is involved in hydrogen bonding with the guanine ring of the nucleotide. Substitution of Asn-128 by 11 other amino acids resulted in a decrease in tetracycline resistance, indicating that tetracycline resistance conferred by Tet(O) is related to GTP binding. The effect of the mutations on the GTP-binding domain is discussed with the EF-Tu-GDP complex as a model. Guanine nucleotide-binding domains share local homolo-gies at the protein sequence level and are similar in their three-dimensional structure and interactions with GTP (2, 7, 11, 12). Many GTP-binding proteins have been identified, and the crystal structures of the two GTPases, EF-Tu and p2lras, have been determined (8, 12). Certain tetracycline resistance (Tcr) determinants have been identified, such as Tet(O) and Tet(M), which also possess the highly conserved sequences of the GTP-binding domain (15, 19, 24). Tet(O