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 (γ-aminobutyric acid) transporter encoded by gabP

Steven C. King, Lisa Brown-Istvan

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

The Escherichia coli GABA (γ-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 (kcat/Km). 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.

Original languageEnglish (US)
Pages (from-to)633-644
Number of pages12
JournalBiochemical Journal
Volume376
Issue number3
DOIs
StatePublished - Dec 15 2003

Keywords

  • Carrier
  • Catalysis
  • Mutagenesis
  • Permease
  • Transport
  • γ-aminobutyric acid (GABA)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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