Mutational analysis of the large periplasmic loop 7-8 of the putrescine transporter PotE in Escherichia coli.
The PotE protein is a putrescine-ornithine antiporter found in many gram-negative bacteria. It is a member of the APA family of transporters and has 12 predicted alpha-helical transmembrane spanning segments (TMS). While the substrate binding site has previously been mapped to a region near the surface of the cytoplasmic lipid layer, no structural feature within the periplasmic domains of PotE have been shown to be important for function. We examined the role of the only large outer loop, situated between transmembrane spanning segment 7 and 8, in putrescine uptake. Deletion of the highly conserved amino acids in the region closest to transmembrane spanning segment 7 produced a protein with little activity. Glycine-scanning mutagenesis of this region showed that Val(249) and Leu(254) were required for optimal transporter function. The V249G mutant transported putrescine at a lower maximal rate compared to wild-type (WT) but with the same substrate binding affinity. In contrast, the L254G mutant had a higher substrate affinity. A series of Val(249) mutants indicated that the hydrophobicity of this residue, which is located at or near the membrane surface, is important for PotE function. Secondary structure predictions of the large outer loop indicated the presence of a hydrophobic alpha-helix in the centre with a hydrophobic region at each end suggesting that the loop was not entirely exposed to the aqueous periplasmic space. The study shows that loop 7-8 is important for PotE function, possibly by forming a re-entrant loop in the channel of the transporter.
Minchin
Rodney F
RF
Centre for Medical Research, University of Western Australia and Laboratory for Cancer Medicine, Western Australian Institute for Medical Research, Royal Perth Hospital, Perth, WA 6000, Australia, rminchin@receptor.pharm.uwa.edu.au
McCoubrie
Joanne E
JE
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry*; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D050356:Lipid Metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011700:Putrescine / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014633:Valine / Q000737:chemistry
D017920:Antiporters; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D011073:Polyamines; C071519:PotE protein, E coli; D007930:Leucine; D014633:Valine; D005998:Glycine; D011700:Putrescine