title: Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B).
abstract: The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4-5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Wright
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: cgt@mrc-lmb.cam.ac.uk.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Tate
mesh_terms: D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013752:Tetracycline / Q000737:chemistry*
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; C079051:tetA protein, Bacteria; D013752:Tetracycline
keywords: Conformational thermostabilisation; Isothermal titration calorimetry; Membrane protein; Transporter