ECD of PepT1 interacts with TM1 to facilitate substrate transport (original) (raw)
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Structure (London, England : 1993), 2015
Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the si...
The International Journal of Biochemistry & Cell Biology, 2009
The mammalian proton-coupled peptide transporter PepT1 is widely accepted as the major route of uptake for dietary nitrogen, as well as being responsible for the oral absorption of a number of classes of drugs, including -lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors. Using sitedirected mutagenesis and zero-trans transport assays, we investigated the role of conserved tyrosines in the transmembrane domains (TMDs) of rabbit PepT1 as predicted by hydropathy plots. All the individual TMD tyrosines were substituted with phenylalanine and shown to retain the ability to traffic to the plasma membrane of Xenopus laevis oocytes. These single substitutions of TMD tyrosines by phenylalanine residues did not affect the proton dependence of peptide uptake, with all retaining wildtype PepT1-like pH dependence. Individual mutations of four of the nine TMD residue tyrosines (Y64, Y287, Y345 and Y587) were without measurable effect on PepT1 function, whereas the other five (Y12, Y56, Y91, Y167 and Y345) were shown to result in altered transport function compared to the wild-type PepT1. Intriguingly, the affinity of Y56F-PepT1 was found to be dramatically increased (approximately 100fold) in comparison to that of the wild-type rabbit PepT1. Y91 mutations also affected the substrate affinity of the transporter, which increased in line with the hydrophilicity of the substituted amino acid (F > Y > Q > R). Y167 was demonstrated to play a pivotal role in rabbit PepT1 function since Y167F, Y167R and Y167Q demonstrated very little transport function. These results are discussed with regard to a proposed mechanism for PepT1 substrate binding.
Communications Chemistry, 2022
Inhibitors for membrane transporters have been shown to be indispensable as drugs and tool compounds. The proton-dependent oligopeptide transporters PEPT1 and PEPT2 from the SLC15 family play important roles in human and mammalian physiology. With Lys[Z(NO2)]-Val (LZNV), a modified Lys-Val dipeptide, a potent transport inhibitor for PEPT1 and PEPT2 is available. Here we present the crystal structure of the peptide transporter YePEPT in complex with LZNV. The structure revealed the molecular interactions for inhibitor binding and a previously undescribed mostly hydrophobic pocket, the PZ pocket, involved in interaction with LZNV. Comparison with a here determined ligand-free structure of the transporter unveiled that the initially absent PZ pocket emerges through conformational changes upon inhibitor binding. The provided biochemical and structural information constitutes an important framework for the mechanistic understanding of inhibitor binding and action in proton-dependent olig...
Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1
European Journal of Biochemistry, 2000
The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH 2), N-acetyl-Phe-amide (Ac-Phe-NH 2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [ 3 H]-d-Phe-l-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH 2 and Ac-Phe-Tyr-NH 2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (K i 1.81^0.37 mm). Ac-Phe-NH 2 had a very weak interaction with PepT1 (K i 16.8^5.64 mm); neither Phe nor Phe-NH 2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (K i 0.10^0.04 mm). The blocked C-terminal peptide Phe-Tyr-NH 2 also interacted with PepT1 with a relatively high affinity (K i 0.94^0.38 mm). Both Ac-Phe-Tyr and Ac-Phe-Tyr-NH 2 interacted weakly with PepT1 (K i 8.41^0.11 and 9.97^4.01 mm, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C-terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.
Journal of Pharmaceutical Sciences, 1998
0 The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di-and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [ 3 H]-(Gly-Sar) was measured. Michaelis−Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFPlabeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced V max .
The mammalian proton-coupled peptide cotransporter PepT1: sitting on the transporter–channel fence?
Philosophical Transactions of the Royal Society B: Biological Sciences, 2009
The proton-coupled di- and tripeptide transporter PepT1 (SLC15a1) is the major route by which dietary nitrogen is taken up from the small intestine, as well as being the route of entry for important therapeutic (pro)drugs such as the β-lactam antibiotics, angiotensin-converting enzyme inhibitors and antiviral and anti-cancer agents. PepT1 is a member of the major facilitator superfamily of 12 transmembrane domain transporter proteins. Expression studies in Xenopus laevis on rabbit PepT1 that had undergone site-directed mutagenesis of a conserved arginine residue (arginine 282 in transmembrane domain 7) to a glutamate revealed that this residue played a role in the coupling of proton and peptide transport and prevented the movement of non-coupled ions during the transporter cycle. Mutations of arginine 282 to other non-positive residues did not uncouple proton–peptide cotransport, but did allow additional ion movements when substrate was added. By contrast, mutations to positive resi...
American Journal of Physiology-Cell Physiology, 2005
The peptide transporter PEPT2 is a polytopic transmembrane protein that mediates the cellular uptake of di- and tripeptides and a variety of peptidomimetics. It is widely expressed in mammalian tissues, including kidney, lung, mammary gland, choroid plexus, and glia cells. In renal tubular cells, PEPT2 is exclusively found at the apical membrane. The molecular mechanisms underlying this polarized expression and targeting to the brush-border membrane are not known. We have explored the role of the 36 COOH-terminal amino acid residues in PEPT2 trafficking and apical expression. EGFP-tagged PEPT2 wild-type transporter and various truncated and mutant proteins were expressed in the polarized proximal tubule cell lines SKPT and OK, and the cellular distribution of the fusion proteins was assessed using confocal microscopy. Whereas deletion of the last seven amino acids (delC7) did not alter PEPT2 surface expression, deletion of the next residue (delC8) or up to 30 terminal amino acids re...
Molecular cloning and tissue distribution of rat peptide transporter PEPT2
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1996
A cDNA encoding rat H+-coupled peptide transporter PEPT2 was isolated. The cDNA encoded a protein of 729 amino acids with 48% amino acid identity to the rat PEPT1. The mRNA expression of rat PEPT2 was predominant in the kidney. When expressed in Xenopus oocytes, rat PEPT2 stimulated the uptake of bestatin, a dipeptide-like drug. The nucleotide sequence data of rat PEPT2 cDNA have been submitted to the DDBJ, EMBL and GenBank nucleotide sequence databases with the following accession number D63149.
Journal of the American Chemical Society
Members of the solute carrier 15 family (SLC15) transport di-and tripeptides as well as peptidomimetic drugs across the cell membrane. Structures of bacterial homologues have provided valuable information on the binding and transport of their natural substrates, but many do not transport medically relevant drugs. In contrast, a homologue from Escherichia coli, DtpA (dipeptide and tripeptide permease), shows a high similarity to human PepT1 (SLC15A1) in terms of ligand selectivity and transports a similar set of drugs. Here, we present the crystal structure of DtpA in ligand-free form (at 3.30 Å resolution) and in complex with the antiviral prodrug valganciclovir (at 2.65 Å resolution) supported by biochemical data. We show that valganciclovir unexpectedly binds with the ganciclovir moiety mimicking the N-terminal residue of a canonical peptide substrate. On the basis of a homology model we argue that this binding mode also applies to the human PepT1 transporter. Our results provide new insights into the binding mode of prodrugs and will assist the rational design of drugs with improved absorption rates.