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J Biol Chem, Vol. 275, Issue 9, 6101-6106, March 3, 2000

Evidence for Interactions between Helices 5 and 8 and a Role for the Interdomain Loop in Tetracycline Resistance Mediated by Hybrid Tet Proteins^*

Cynthia A. Saraceni-Richards and Stuart B. Levy

From the Center for Adaptation Genetics and Drug Resistance and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111

An interdomain hybrid Tet protein consisting of a class C  domain and a class B  domain (Tet(C/B)) lacks detectable efflux ability and provides only minimal levels of resistance to tetracycline (Tc) (3 µg/ml) compared with intact class B (256 µg/ml) and class C (64 µg/ml). Twenty-one independently isolated mutants of the Tet(C/B) protein with increased Tc resistance were generated by random chemical mutagenesis. Nine mutants with a Glu substitution for Gly-152 in helix 5 of the class C  domain produced a resistance of 48 µg/ml, whereas another 9 with an Asp replacement of Gly-247 in helix 8 of the class B  domain mediated resistance at 32 µg/ml. The third type of mutation, found in 3 mutants expressing 24 µg/ml resistance, was a S202F replacement in the putative interdomain cytoplasmic loop of Tet(C/B). The latter underscores a previously unappreciated function of the interdomain cytoplasmic loop. All three types of Tet(C/B) mutant proteins were expressed in amounts comparable with that of the original protein and demonstrated restored energy-dependent efflux of tetracycline. Site-directed mutational analysis demonstrated that a Gly-247 to Asn mutation could also facilitate Tc resistance by the Tet(C/B) hybrid, and a negatively charged side chain at position 152 was required for Tet(C/B) activity. These mutations appear to promote the necessary functional interactions between the interclass domains that do not occur in the Tet(C/B) hybrid protein and suggest a direct association between helix 5 and helix 8 in the function of Tet efflux proteins.

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