Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters.
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ABSTRACT:
SUBMITTER: Lyons JA
PROVIDER: S-EPMC4149780 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A major challenge in drug development is the optimization of intestinal absorption and cellular uptake. A successful strategy has been to develop prodrug molecules, which hijack solute carrier (SLC) transporters for active transport into the body. The proton-coupled oligopeptide transporters, PepT1 and PepT2, have been successfully targeted using this approach. Peptide transporters display a remarkable capacity to recognize a diverse library of di- and tripeptides, making them extremely promiscuous and major contributors to the pharmacokinetic profile of several important drug classes, including beta-lactam antibiotics and antiviral and antineoplastic agents. Of particular interest has been their ability to recognize amino acid and peptide-based prodrug molecules, thereby providing a rational approach to improving drug transport into the body. However, the structural basis for prodrug recognition has remained elusive. Here we present crystal structures of a prokaryotic homolog of the mammalian transporters in complex with the antiviral prodrug valacyclovir and the peptide-based photodynamic therapy agent, 5-aminolevulinic acid. The valacyclovir structure reveals that prodrug recognition is mediated through both the amino acid scaffold and the ester bond, which is commonly used to link drug molecules to the carrier's physiological ligand, whereas 5-aminolevulinic acid makes far fewer interactions compared with physiological peptides. These structures provide a unique insight into how peptide transporters interact with xenobiotic molecules and provide a template for further prodrug development.
| S-EPMC6338836 | biostudies-literature
Project description:BACKGROUND:Cellular uptake of small peptides is an important physiological process mediated by the PTR family of proton-coupled peptide transporters. In bacteria peptides can be used as a source of amino acids and nitrogen. Similarly in humans peptide transport is the principle route for the uptake and retention of dietary protein in the form of short di- and tri-peptides for cellular metabolism. SCOPE OF THE REVIEW:Recent crystal structures of bacterial PTR family transporters, combined with biochemical studies of transport have revealed key molecular details underpinning ligand promiscuity and the mechanism of proton-coupled transport within the family. MAJOR CONCLUSIONS:Pairs of salt bridge interactions between transmembrane helices work in tandem to orchestrate alternating access transport within the PTR family. Key roles for residues conserved between bacterial and eukaryotic homologues suggest a conserved mechanism of peptide recognition and transport that in some cases has been subtly modified in individual species. GENERAL SIGNIFICANCE:Physiological studies on PepT1 and PepT2, the mammalian members of this family, have identified these transporters as being responsible for the uptake of many pharmaceutically important drug molecules, including antibiotics and antiviral medications and demonstrated their promiscuity can be used for improving the oral bioavailability of poorly absorbed compounds. The insights gained from recent structural studies combined with previous physiological and biochemical analyses are rapidly advancing our understanding of this medically important transporter superfamily. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.
| S-EPMC4331665 | biostudies-literature
Project description:Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.
| S-EPMC3419923 | biostudies-literature
Project description:Short-chain peptides are transported across membranes through promiscuous proton-dependent oligopeptide transporters (POTs)--a subfamily of the major facilitator superfamily (MFS). The human POTs, PEPT1 and PEPT2, are also involved in the absorption of various drugs in the gut as well as transport to target cells. Here, we present a structure of an oligomeric POT transporter from Shewanella oneidensis (PepTSo2), which was crystallized in the inward open conformation in complex with the peptidomimetic alafosfalin. All ligand-binding residues are highly conserved and the structural insights presented here are therefore likely to also apply to human POTs.
| S-EPMC3790050 | biostudies-literature
Project description: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.
| S-EPMC4705975 | biostudies-literature
Project description:Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as ?-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.
| S-EPMC3710879 | biostudies-literature
Project description:Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K+/H+ symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins.
| S-EPMC6994465 | biostudies-literature
Project description:Mammalian members of the proton-coupled oligopeptide transporter family (SLC15) are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs. The driving force for uphill electrogenic symport is the chemical gradient and membrane potential which favors proton uptake into the cell along with the peptide/mimetic substrate. The peptide transporters are responsible for the absorption and conservation of dietary protein digestion products in the intestine and kidney, respectively, and in maintaining homeostasis of neuropeptides in the brain. They are also responsible for the absorption and disposition of a number of pharmacologically important compounds including some aminocephalosporins, angiotensin-converting enzyme inhibitors, antiviral prodrugs, and others. In this review, we provide updated information on the structure-function of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4) and PhT2 (SLC15A3), and their expression and localization in key tissues. Moreover, mammalian peptide transporters are discussed in regard to pharmacogenomic and regulatory implications on host pharmacology and disease, and as potential targets for drug delivery. Significant emphasis is placed on the evolving role of these peptide transporters as elucidated by studies using genetically modified animals. Whenever possible, the relevance of drug-drug interactions and regulatory mechanisms are evaluated using in vivo studies.
| S-EPMC3602806 | biostudies-literature
Project description:POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.
| S-EPMC5740623 | biostudies-literature
Project description:A group of OPTs (oligopeptide transporters) exclusively identified in plants and fungi are proposed to transport oligopeptides and derivatives of three to six amino acids in length, but their transport mechanisms and biological functions are poorly understood. We expressed the Saccharomyces cerevisiae (yeast) OPT ScOPT1 and five Arabidopsis thaliana AtOPTs in Xenopus laevis oocytes for two-electrode voltage-clamp studies. ScOPT1 produced inward currents in response to GSH or GSSG, the phytochelatin (PC) PC2 and oligopeptides including the tetrapeptide GGFL, but not KLGL. Inward currents were dependent on the external proton and substrate concentrations, with high affinity for both. This and the inward currents evoked by substrates with net negative charges showed that ScOPT1 is a proton-coupled transporter. ScOPT1 displayed highest apparent affinity for PC2, with small differences in the maximal current among substrates. Glutathione transport by any of the tested AtOPTs, including AtOPT6, was not detected in yeast growth complementation assays. With AtOPT4, initially only small KLGL-dependent currents were recorded in batches of oocytes showing high ScOPT1 expression. AtOPT4 expression was optimized by swapping the 5'-untranslated region with that of ScOPT1. AtOPT4 displayed a higher affinity for KLGL than ScOPT1 did for any peptide. AtOPT4-mediated KLGL transport was detectable at pH 5.0, but not at pH 6.0 or 7.0. Taken together, our results demonstrate that ScOPT1 and AtOPT4 are proton-coupled OPTs with broad but distinct substrate specificities and affinities.
| S-EPMC1383685 | biostudies-other