Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1.
The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.
Weitz
Dietmar
D
Molecular Nutrition Unit, Department of Food and Nutrition, Technical University of Munich, 85350 Freising, Germany.
Harder
Daniel
D
Casagrande
Fabio
F
Fotiadis
Dimitrios
D
Obrdlik
Petr
P
Kelety
Bela
B
Daniel
Hannelore
H
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008322:Mammals; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics*; D000074011:Peptide Transporter 1; D016133:Polymerase Chain Reaction; D011510:Proteolipids / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000074011:Peptide Transporter 1; D011510:Proteolipids; D027981:Symporters; C518647:dtpA protein, E coli; C020485:proteoliposomes