title: Evidence for interactions between helices 5 and 8 and a role for the interdomain loop in tetracycline resistance mediated by hybrid Tet proteins.
abstract: An interdomain hybrid Tet protein consisting of a class C alpha domain and a class B beta domain (Tet(C/B)) lacks detectable efflux ability and provides only minimal levels of resistance to tetracycline (Tc) (3 microg/ml) compared with intact class B (256 microg/ml) and class C (64 microg/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 alpha domain produced a resistance of 48 microg/ml, whereas another 9 with an Asp replacement of Gly-247 in helix 8 of the class B beta domain mediated resistance at 32 microg/ml. The third type of mutation, found in 3 mutants expressing 24 microg/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.
authors.affiliation: Center for Adaptation Genetics and Drug Resistance and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Saraceni-Richards
authors.forename: S B
authors.initials: SB
authors.lastname: Levy
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline