Functional Characterization of the Putative POT from Clostridium perfringens.

Proton-coupled oligopeptide transporters (POTs) are a fundamental part of the cellular transport machinery that provides plants, bacteria, and mammals with nutrition in the form of short peptides. However, POTs are not restricted to peptide transport; mammalian POTs have especially been in focus due to their ability to transport several peptidomimetics in the small intestine. Herein, we studied a POT from Clostridium perfringens (CPEPOT), which unexpectedly exhibited atypical characteristics. First, very little uptake of a fluorescently labelled peptide ß-Ala-Lys-AMCA, an otherwise good substrate of several other bacterial POTs, was observed. Secondly, in the presence of a competitor peptide, enhanced uptake of ß-Ala-Lys-AMCA was observed due to trans-stimulation. This effect was also observed even in the absence of a proton electrochemical gradient, suggesting that ß-Ala-Lys-AMCA uptake mediated by CPEPOT is likely through the substrate-concentration-driving exchange mechanism, unlike any other functionally characterized bacterial POTs.

Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1.

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10.

Interleukin-10 (IL-10) is an immunoregulatory cytokine with both anti-inflammatory and immunostimulatory properties and is frequently dysregulated in disease. We used a structure-based approach to deconvolute IL-10 pleiotropy by determining the structure of the IL-10 receptor (IL-10R) complex by cryo-electron microscopy at a resolution of 3.5 angstroms. The hexameric structure shows how IL-10 and IL-10Rα form a composite surface to engage the shared signaling receptor IL-10Rß, enabling the design of partial agonists. IL-10 variants with a range of IL-10Rß binding strengths uncovered substantial differences in response thresholds across immune cell populations, providing a means of manipulating IL-10 cell type selectivity. Some variants displayed myeloid-biased activity by suppressing macrophage activation without stimulating inflammatory CD8+ T cells, thereby uncoupling the major opposing functions of IL-10. These results provide a mechanistic blueprint for tuning the pleiotropic actions of IL-10.

Molecular architecture of the Jumonji C family histone demethylase KDM5B.

The full length human histone 3 lysine 4 demethylase KDM5B (PLU-1/Jarid1B) has been studied using Hydrogen/Deuterium exchange mass spectrometry, homology modelling, sequence analysis, small angle X-ray scattering and electron microscopy. This first structure on an intact multi-domain Jumonji histone demethylase reveal that the so-called PLU region, in the central region of KDM5B, has a curved α-helical three-dimensional structure, that acts as a rigid linker between the catalytic core and a region comprising four α-helices, a loop comprising the PHD2 domain, two large intrinsically disordered loops and the PHD3 domain in close proximity. The dumbbell shaped and curved KDM5B architecture observed by electron microscopy is complementary to the nucleosome surface and has a striking overall similarity to that of the functionally related KDM1A/CoREST complex. This could suggest that there are similarities between the demethylation mechanisms employed by the two histone 3 lysine 4 demethylases at the molecular level.

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state.

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl ß-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.

The prototypical proton-coupled oligopeptide transporter YdgR from Escherichia coli facilitates chloramphenicol uptake into bacterial cells.

Chloramphenicol (Cam) is a broad-spectrum antibiotic used to combat bacterial infections in humans and animals. Cam export from bacterial cells is one of the mechanisms by which pathogens resist Cam's antibacterial effects, and several different proteins are known to facilitate this process. However, to date no report exists on any specific transport protein that facilitates Cam uptake. The proton-coupled oligopeptide transporter (POT) YdgR from Escherichia coli is a prototypical member of the POT family, functioning in proton-coupled uptake of di- and tripeptides. By following bacterial growth and conducting LC-MS-based assays we show here that YdgR facilitates Cam uptake. Some YdgR variants displaying reduced peptide uptake also exhibited reduced Cam uptake, indicating that peptides and Cam bind YdgR at similar regions. Homology modeling of YdgR, Cam docking, and mutational studies suggested a binding mode that resembles that of Cam binding to the multidrug resistance transporter MdfA. To our knowledge, this is the first report of Cam uptake into bacterial cells mediated by a specific transporter protein. Our findings suggest a specific bacterial transporter for drug uptake that might be targeted to promote greater antibiotic influx to increase cytoplasmic antibiotic concentration for enhanced cytotoxicity.

Several hPepT1-transported drugs are substrates of the Escherichia coli proton-coupled oligopeptide transporter YdgR.

Proton-dependent oligopeptide transporters (POTs) are secondary active transporters found in all kingdoms of life. POTs utilize the proton electrochemical gradient for the uptake of nutrient dipeptides and tripeptides. The human POT hPepT1 is known to transport a number of drugs. As part of ongoing studies on substrate specificities of POTs from Escherichia coli, our aim in this study was to investigate whether bacterial POTs could also transport these drugs. For this, we selected the common orally administered drugs sulpiride, bestatin, valacyclovir, ampicillin and oseltamivir, that are all transported by hPepT1. The transport of these drugs was evaluated using the prototypical POT YdgR from E. coli. The transport studies were pursued through combining cell-based assays with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These investigations revealed that YdgR from E. coli is able to transport five (sulpiride, bestatin, valacyclovir, ampicillin and oseltamivir) drugs. Furthermore, cells not overexpressing YdgR were also able to transport these drugs in a POT-like manner. Orthologues of YdgR are found in several species in the gut microbiome; hence, our findings could have implications for further understanding about the interaction between gut microbes and orally administered drugs.

Peptide Selectivity of the Proton-Coupled Oligopeptide Transporter from Neisseria meningitidis.

Peptide transport in living organisms is facilitated by either primary transport, hydrolysis of ATP, or secondary transport, cotransport of protons. In this study, we focused on investigating the ligand specificity of the Neisseria meningitidis proton-coupled oligopeptide transporter (NmPOT). It has been shown that the gene encoding this transporter is upregulated during infection. NmPOT conformed to the typical chain length preference as observed in prototypical transporters of this family. In contrast to prototypical transporters, it was unable to accommodate a positively charged peptide residue at the C-terminus position of the substrate peptide. Sequence analysis of the active site of NmPOT displayed a distinctive aromatic patch, which has not been observed in any other transporters from this family. This aromatic patch may be involved in providing NmPOT with its atypical preferences. This study provides important novel information towards understanding how these transporters recognize their substrates.

Salt Bridge Swapping in the EXXERFXYY Motif of Proton-coupled Oligopeptide Transporters.

Proton-coupled oligopeptide transporters (POTs) couple the inward transport of di- or tripeptides with an inwardly directed transport of protons. Evidence from several studies of different POTs has pointed toward involvement of a highly conserved sequence motif, E1XXE2RFXYY (from here on referred to as E1XXE2R), located on Helix I, in interactions with the proton. In this study, we investigated the intracellular substrate accumulation by motif variants with all possible combinations of glutamate residues changed to glutamine and arginine changed to a tyrosine, the latter being a natural variant found in the Escherichia coli POT YjdL. We found that YjdL motif variants with E1XXE2R, E1XXE2Y, E1XXQ2Y, or Q1XXE2Y were able to accumulate peptide, whereas those with E1XXQ2R, Q1XXE2R, or Q1XXQ2Y were unable to accumulate peptide, and Q1XXQ2R abolished uptake. These results suggest a mechanism that involves swapping of an intramotif salt bridge, i.e. R-E2 to R-E1, which is consistent with previous structural studies. Molecular dynamics simulations of the motif variants E1XXE2R and E1XXQ2R support this mechanism. The simulations showed that upon changing conformation arginine pushes Helix V, through interactions with the highly conserved FYING motif, further away from the central cavity in what could be a stabilization of an inward facing conformation. As E2 has been suggested to be the primary site for protonation, these novel findings show how protonation may drive conformational changes through interactions of two highly conserved motifs.

Critical role of a conserved transmembrane lysine in substrate recognition by the proton-coupled oligopeptide transporter YjdL.

Proton-coupled oligopeptide transporters (POTs) utilize an electrochemical proton gradient to accumulate peptides in the cytoplasm. Changing the highly conserved active-site Lys117 in the Escherichia coli POT YjdL to glutamine resulted in loss of ligand affinity as well as inability to distinguish between a dipeptide ligand and the corresponding dipeptide amide. The radically changed pH(Bulk) profiles of Lys117Gln and Lys117Arg mutants indicate an important role of Lys117 in facilitating protonation of the transporter; a notion that is supported by the close proximity of Lys117 to the conserved ExxERFxYY POT motif previously shown to be involved in proton translocation. These results point toward a novel dual role of Lys117 in direct or indirect interaction with both proton and peptide.

New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay.

Proton-coupled oligopeptide transporters (POTs) are secondary active transporters that facilitate di- and tripeptide uptake by coupling it to an inward directed proton electrochemical gradient. Here the substrate specificities of Escherichia coli POTs YdgR, YhiP and YjdL were investigated by means of a label free transport assay using the hydrophilic pH sensitive dye pyranine and POT overexpressing E. coli cells. The results confirm and extend the functional knowledge on E. coli POTs. In contrast to previous assumptions, alanine and trialanine appears to be substrates of YjdL, albeit poor compared to dipeptides. Similarly tetraalanine apparently is a substrate of both YdgR and YhiP.