Induction of substrate specificity shifts by placement of alanine insertions within 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 is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic [SHS (sensitive hydrophobic surface)] and the other hydrophilic [SPS (sensitive polar surface)]. To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed. Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state [(3)H]GABA accumulation. TSR (transport specificity ratio) analysis, employing [(3)H]nipecotic acid and [(14)C]GABA, showed that certain alanine insertions were associated with a specificity shift (i.e. a change in k (cat)/ K (m)). An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for [(3)H]nipecotic acid relative to [(14)C]GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect. Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity ( k (cat)/ K (m)) phenotype. The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E. coli phenylalanine permease PheP [Pi, Chow and Pittard (2002) J. Bacteriol. 184, 5842-5847] that have a functionally significant CAR-like domain.
King
Steven C
SC
Department of Integrated Biosciences, Oregon Health & Science University, Portland, OR 97239-3097, USA. kingst@ohsu.edu
Hu
Liaoyuan A
LA
Pugh
Amy
A
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050485:GABA Plasma Membrane Transport Proteins; D017930:Genes, Reporter; D057927:Hydrophobic and Hydrophilic Interactions; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D027361:Organic Anion Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; 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; D027361:Organic Anion Transporters; C513601:gabP protein, E coli; D005680:gamma-Aminobutyric Acid; D001616:beta-Galactosidase; D000409:Alanine