Functional significance of the "signature cysteine" in helix 8 of the Escherichia coli 4-aminobutyrate transporter from the amine-polyamine-choline superfamily. Restoration of Cys-300 to the Cys-less Gabp.
gab permease (GabP) is the exclusive mediator of 4-aminobutyrate (GABA) transport across the Escherichia coli plasma membrane. Helix 8 and a portion of the adjoining cytoplasmic region (loop 8-9) constitute the GabP "consensus amphipathic region" (CAR), a potential channel-forming domain that is found to be evolutionarily conserved within the APC (amine-polyamine-choline) transporter superfamily. Upon the polar surface of the CAR, all known gab permeases display a "signature cysteine" not found in other members of the APC superfamily, suggesting that discrete features within the CAR might play a role in imparting specificity (kcat/Km) to the translocation reaction. Here we show that among the five cysteine residues in the E. coli GabP, only Cys-300, the signature cysteine, can restore wild type properties to the Cys-less GabP mutant. We conclude (i) from partial reaction studies (equilibrium exchange, counterflow) that rapid translocation of the GABA binding site from one side of the membrane to the other is greatly facilitated by Cys-300 and (ii) from pharmacological studies that loss of Cys-300 has little effect on the affinity that GabP exhibits for a structurally diverse array (kojic amine, 5-aminovaleric acid, GABA, nipecotic acid, and cis-4-aminocrotonic acid) of competitive ligands. These results raise the possibility that other GABA transporters might rely analogously upon conserved cysteine residues positioned within the amphipathic helix 8 and loop 8-9 regions.
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Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641, USA.
King
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry*; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters; D016415:Sequence Alignment; D005680:gamma-Aminobutyric Acid / Q000493:pharmacokinetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C513601:gabP protein, E coli; D005680:gamma-Aminobutyric Acid; D003545:Cysteine