Functional and Structural Characterization of a Prokaryotic Peptide Transporter with Features Similar to Mammalian PEPT1

Abstract

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 ϳ8 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.

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT

“…S8A). Alafosfalin is a good substrate for POT family transporters (29)(30)(31) and competes with the uptake of the (Ala) 2 peptide by GkPOT ( Fig. 2A).…”

Bacterial peptide transporters: Messengers of nutrition to virulence

“…Similarly, the presence of tyrosine and tryptophan residues on specific transmembrane domains and FWALF motif in TM7 affect the V max and K m of the transport of peptides. 27,35,36 As proven by the study on YdgR peptide transporter of E. coli, 38 PTR transporters function in monomeric form. Several residues were found to be significant for binding of proton such as arginine, tyrosine and glutamine in TM1, lysine in TM4 and glutamine in TM7 and TM10.…”

“…60 Similar studies are carried out by expressing bacterial transporters in laboratory bacterial strains like E. coli BL21. 38 Thus, with the help of these methodologies it is possible to characterize many unknown peptide transporters from the genome of pathogenic bacteria.…”

“…27 The transport of peptides by PTR transporters depends on the ionization state of the peptides where the inward electrochemical gradient of protons provides the energy for transport. 37,38 The stoichiometry remains as one proton for cationic or neutral dipeptide, 2 protons for anionic dipeptide and 3 protons for anionic tripeptide, where the extra protons neutralize the charge on the peptide. 29,39,40 Conserved sequences on the transmembrane domains allow specific binding with the peptides and thereby effective transport, like the ExxERFxYY motif on the transmembrane helix 1 (TM1) facilitates hydrogen bond formation with the carboxy terminus of the dipeptide.…”

“…99 Direct methods like microscopic examination of fluorescently labeled peptides taken by bacterial cells, 13 use of radiolabeled substrates 68 and chromatography mediated analysis of the peptides present inside the cell 100 would describe the function of the transporter more specifically. Use of proton ionophores 38 or measuring alkalization as a result of proton translocation would be helpful to study PTR peptide transporters. 26 Mammalian peptide transporters have been exogenously expressed in Xenopus oocytes to characterize their function and determine the nature of peptide substrates.…”