title: Functional investigation of conserved membrane-embedded glutamate residues in the proton-coupled peptide transporter YjdL.
abstract: Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ~0.1 to ~1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC50 increased to ~2.0 and > 10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.
authors.affiliation: Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Johanne M
authors.initials: JM
authors.lastname: Jensen
authors.forename: Heidi  A
authors.initials: H
authors.lastname: Ernst
authors.forename: Xiaole
authors.initials: X
authors.lastname: Wang
authors.forename: Helle
authors.initials: H
authors.lastname: Hald
authors.forename: Amarah C
authors.initials: AC
authors.lastname: Ditta
authors.forename: Fouzia
authors.initials: F
authors.lastname: Ismat
authors.forename: Moazur
authors.initials: M
authors.lastname: Rahman
authors.forename: Osman
authors.initials: O
authors.lastname: Mirza
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D011522:Protons
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D011522:Protons; C544083:YjdL protein, E coli; D018698:Glutamic Acid