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.

BackgroundPeptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood.ResultsThe X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1’s substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter.ConclusionThis study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0167-8) contains supplementary material, which is available to authorized users.

Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 ؎ 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acidcontaining outer membranes.Mycobacteria are classified as Gram-positive bacteria but have evolved a complex cell wall, comprising a peptidoglycanarabinogalactan polymer with covalently bound mycolic acids of considerable length (up to 90 carbon atoms) and a large variety of extractable lipids (1, 2). Most of these lipids are constituents of the cell envelope that provides an extraordinarily efficient permeability barrier and is an essential part of the intrinsic resistance of mycobacteria to many toxic compounds and antibiotics (3). To account for these observations, Minnikin (4) proposed a model in which the mycolic acids form the inner leaflet of an asymmetrical bilayer. Mutants and treatments affecting mycolic acid biosynthesis and the production of extractable lipids showed an increase in cell wall permeability and a drastic decrease in virulence, underlining the importance of the cell wall integrity for intracellular survival of Mycobacterium tuberculosis (1). Cryoelectron tomography revealed the native organization of the Mycobacterium smegmatis cell envelope. Further, the three-dimensional data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, M. bovis BCG, and Corynebacterium glutamicum identified the outermost layer as a lipid bilayer. Mycolic acids were shown to be essential components of this bilayer, therefore providing the first visualization of mycobacterial outer membranes in their native state (5).These findings raise the question of how the mycobacterial outer membrane is functionalized fo...

Graphical abstract

Uptake of peptides into Escherichia coli cells is thought to be mediated by three different transport systems represented by the dipeptide permease Dpp, the tripeptide permease TppB, and the oligopeptide permease Opp [1][2][3]. Despite the fact that these proteins seem to discriminate according to the backbone length of peptide substrates, they do show some overlapping specificity. Although their prime physiological role is in the uptake of peptide-bound amino acids as an economic process to provide energy substrates and building blocks for cellular metabolism, peptide uptake seems also to be involved in signaling processes and metabolic adaptation [4]. Whereas Opp and Dpp represent ATP-binding cassette transporters with periplasmatic binding proteins [5][6][7], TppB belongs to the family of proton-dependent peptide symporters that utilize the proton gradient as driving force and lack cognate binding proteins [8]. After identification of the The genome of Escherichia coli contains four genes assigned to the peptide transporter (PTR) family. Of these, only tppB (ydgR) has been characterized, and named tripeptide permease, whereas protein functions encoded by the yhiP, ybgH and yjdL genes have remained unknown. Here we describe the overexpression of yhiP as a His-tagged fusion protein in E. coli and show saturable transport of glycyl-sarcosine (Gly-Sar) with an apparent affinity constant of 6.5 mm. Overexpression of the gene also increased the susceptibility of cells to the toxic dipeptide alafosfalin. Transport was strongly decreased in the presence of a protonophore but unaffected by sodium depletion, suggesting H + -dependence. This was confirmed by purification of YhiP and TppB by nickel affinity chromatography and reconstitution into liposomes. Both transporters showed Gly-Sar influx in the presence of an artificial proton gradient and generated transport currents on a chip-based sensor. Competition experiments established that YhiP transported dipeptides and tripeptides. Western blot analysis revealed an apparent mass of YhiP of 40 kDa. Taken together, these findings show that yhiP encodes a protein that mediates proton-dependent electrogenic transport of dipeptides and tripeptides with similarities to mammalian PEPT1. On the basis of our results, we propose to rename YhiP as DtpB (dipeptide and tripeptide permease B), by analogy with the nomenclature in other bacteria. We also propose to rename TppB as DtpA, to better describe its function as the first protein of the PTR family characterized in E. coli.Abbreviations AMCA, b-Ala-Lys-N

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.

For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system.