![]() ![]() A range of modified dipeptides that specifically inhibit mammalian peptide transporters have been developed 47, 48, 49, 50, 51. Thorough investigation of transport processes by membrane proteins is facilitated by the availability of specific high-affinity inhibitors. Cryo-electron microscopy studies provided now also outward-open and outward-facing occluded states with the structures of mammalian PEPT1 45 and PEPT2 45, 46. Until recently, all structures of POTs were either in the inward-open or inward-facing occluded conformation 3. As other MFS transporters, POTs translocate their substrates using an alternate access mechanism, involving outward-open and outward-facing occluded as well as inward-open and inward-facing occluded states 6, 7, 9. It was proposed that the multispecificity observed in POTs arises in part due to water network modulations and movements of (predominantly aromatic) residues in those pockets 39. Furthermore, hydrophobic pockets accommodating the N-terminal (P1) and C-terminal (P2) amino acid side chains of dipeptides 38, 39, as well as one accommodating the additional side chain in tripeptides 40 (P3), were described. Although variations in the binding mechanism of different ligands were observed 39, 40, 43, several conserved binding pocket residues were identified, which interact with the peptide/peptidomimetic backbone of most ligands 44. Over the last years, several crystal structures of bacterial homologues were solved in the apo form 28, 29, 30, 31, 32, 33, 34, 35, 36 and with bound dipeptides 37, 38, 39, tripeptides 37, 38, 40 and other peptidomimetic ligands 30, 36, 41, 42, 43. Recently, inhibition of PEPT1 in inflammatory bowel disease patients was proposed as therapeutic approach to block membrane transport of toxic bacterial products 27. PEPT1 and PEPT2 have pharmacological relevance, as they were shown to translocate a wide range of peptidomimetic drugs and prodrugs 20 such as antibiotics 21, 22, 23, 24 (e.g., cefadroxil and cefprozil), antivirals 25 (e.g., valaciclovir), protease inhibitors 26 (e.g., bestatin) or Parkinson medications 25 (e.g., l-dopa- l-Phe, L-dopa). Both transporters are also expressed in other human and mammalian cell types 18. While PEPT1, a high-capacity, low-affinity transporter mainly expressed in the intestine, takes up dietary di- and tripeptides, PEPT2, a low-capacity, high-affinity transporter is predominantly expressed in the kidney, where it is responsible for the reabsorption of di- and tripeptides 18. In humans, the POTs PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have important physiological and pharmacological roles 18, 19. Valuable information on the transport function of POTs was obtained from bacterial family members, e.g., from the well-studied peptide transporters from the bacterium Escherichia coli 10, 11, 12, 13, 14, 15, 16, 17. They actively transport and accumulate their substrates across cellular membranes using the proton electrochemical gradient 3. The proton-dependent oligopeptide transporter (POT) family 1, 2, 3, 4 (also referred to as the peptide transporter (PTR) family 2, 5) is a group of secondary active symporters 6, 7, which belong to the major facilitator superfamily (MFS) 8, 9. The provided biochemical and structural information constitutes an important framework for the mechanistic understanding of inhibitor binding and action in proton-dependent oligopeptide transporters. 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 structure revealed the molecular interactions for inhibitor binding and a previously undescribed mostly hydrophobic pocket, the PZ pocket, involved in interaction with LZNV. Here we present the crystal structure of the peptide transporter YePEPT in complex with LZNV. With Lys-Val (LZNV), a modified Lys-Val dipeptide, a potent transport inhibitor for PEPT1 and PEPT2 is available. The proton-dependent oligopeptide transporters PEPT1 and PEPT2 from the SLC15 family play important roles in human and mammalian physiology. Inhibitors for membrane transporters have been shown to be indispensable as drugs and tool compounds. ![]()
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