Use of the transport specificity ratio and cysteine-scanning mutagenesis to detect multiple substrate specificity determinants in the consensus amphipathic region of the Escherichia coli GABA (gamma-aminobutyric acid) transporter encoded by gabP.
The Escherichia coli GABA (gamma-aminobutyric acid) permease, GabP, and other members of the APC (amine/polyamine/choline) transporter superfamily share a CAR (consensus amphipathic region) that probably contributes to solute translocation. If true, then the CAR should contain structural features that act as determinants of substrate specificity ( k (cat)/ K (m)). In order to address this question, we have developed a novel, expression-independent TSR (transport specificity ratio) analysis, and applied it to a series of 69 cysteine-scanning (single-cysteine) variants. The results indicate that GabP has multiple specificity determinants (i.e. residues at which an amino acid substitution substantially perturbs the TSR). Specificity determinants were found: (i) on a hydrophobic surface of the CAR (from Leu-267 to Ala-285), (ii) on a hydrophilic surface of the CAR (from Ser-299 to Arg-318), and (iii) in a cytoplasmic loop (His-233) between transmembrane segments 6 and 7. Overall, these observations show that (i) structural features within the CAR have a role in substrate discrimination (as might be anticipated for a transport conduit) and, interestingly, (ii) the substrate discrimination task is shared among specificity determinants that appear too widely dispersed across the GabP molecule to be in simultaneous contact with the substrates. We conclude that GabP exhibits behaviour consistent with a broadly applicable specificity delocalization principle, which is demonstrated to follow naturally from the classical notion that translocation occurs synchronously with conformational transitions that change the chemical potential of the bound ligand [Tanford (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2882-2884].
King
Steven C
SC
Department of Integrative Biosciences, Oregon Health & Science University, Portland, OR 97239-3097, USA. kingst@ohsu.edu
Brown-Istvan
Lisa
L
D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence; D003545:Cysteine / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050485:GABA Plasma Membrane Transport Proteins; D057927:Hydrophobic and Hydrophilic Interactions; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009557:Nipecotic Acids / Q000378:metabolism; D027361:Organic Anion Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009557:Nipecotic Acids; D027361:Organic Anion Transporters; C513601:gabP protein, E coli; C030278:nipecotic acid; D007544:Isopropyl Thiogalactoside; D005680:gamma-Aminobutyric Acid; D003545:Cysteine