Peptide transporter structure reveals binding and action mechanism of a potent PEPT1 and PEPT2 inhibitor

Abstract

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 oligopeptide transporters.

Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition

“…DtpB adopts the canonical MFS fold, characterized by two six transmembrane helix bundles. The N-and C-terminal bundles are linked by the HA-HB helices, as observed in other bacterial POTs [3][4][5][6][7][8]10,18,[32][33][34][35]37,39 . The overall structure of DtpB is similar to DtpA, DtpC, and DtpD with Cα atom RMSD (root-mean-square deviation) values of 0.9, 1.3, and 1.2 Å, respectively.…”

“…In the last decade, over 50 entries of the POT family were deposited in the Protein Data Bank (PDB), representing eleven bacterial and two mammalian homologs, bound to eight unique natural di-and tripeptides (Ala-Phe, Ala-Glu, Ala-Gln, Ala-Leu, Phe-Ala, Phe-Ala-Gln, Ala-Ala-Ala, alafosfalin), peptidomimetic drugs (valaciclovir, valganciclovir, 5-aminolevulinic acid) and a potent transport inhibitor Lys[Z(NO2)]-Val [3][4][5][6][7][8]10,18,[31][32][33][34][35][36][37][38][39][40][41] . These efforts revealed the basic principle underlying peptide binding in POTs which can be described as an electrostatic clamping mechanism between the invariable part of peptides (N-and C-termini as well as the peptide backbone) and conserved residues in the transporter (mainly via arginine, lysine, glutamate, asparagine and tyrosine residues).…”

The mechanism of mammalian proton-coupled peptide transporters

“…( Newstead et al, 2011 ) They operate via an alternating access mechanism encoded in four inverted topology repeats, progressively reorienting the N- and C-terminal bundles to cycle through outwards-facing (OF), occluded (OCC) and inwards-facing (IF) states. ( Radestock and Forrest, 2011 ) Since the first structure of a POT family member was published ( Newstead et al, 2011 ), many procaryotic ( Solcan et al, 2012; Guettou et al, 2013; Doki et al, 2013; Lyons et al, 2014; Guettou et al, 2014; Zhao et al, 2014; Fowler et al, 2015; Boggavarapu et al, 2015; Beale et al, 2015; Parker et al, 2017; Martinez Molledo et al, 2018; Ural-Blimke et al, 2019; Minhas and Newstead, 2019; Stauffer et al, 2022; Kotov et al, 2023 ) and plant ( Parker and Newstead, 2014; Sun et al, 2014 ) homologues have been structurally and biochemically characterised, all in IF states with varying degrees of occlusion (see figure 1a for an overview of available POT structures and their conformational states). Several residues have been suggetsed to be involved in proton transfer, including a partially conserved histidine on TM2 (H87; residue numbers refer to PepT2, if not specified otherwise) ( Terada et al, 1996; Fei et al, 1997; Chen et al, 2000; Omori et al, 2021; Parker et al, 2021 ) and two conserved glutamates on TM1 (E53 and E56) ( Jensen et al, 2012; Doki et al, 2013; Aduri et al, 2015 ), while simulations have helped our understanding of proton-transfer processes and conformational changes.…”

Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C

“…POTs have been intensively studied on a structural and biochemical level over the last 30 years. More than 50 entries for this transporter class can be found in the protein data bank, representing ten different bacterial homologues and the mammalian PepT1 and PepT2 transporters, bound to a limited set of substrates and drugs ( Newstead et al, 2011 ; Solcan et al, 2012 ; Doki et al, 2013 ; Guettou et al, 2013 ; Guettou et al, 2014 ; Lyons et al, 2014 ; Zhao et al, 2014 ; Quistgaard et al, 2017 ; Martinez Molledo et al, 2018a ; Martinez Molledo et al, 2018b ; Minhas et al, 2018 ; Nagamura et al, 2019 ; Ural-Blimke et al, 2019 ; Killer et al, 2021 ; Parker et al, 2021 ; Shen et al, 2022 ; Stauffer et al, 2022 ). Although bacterial and eukaryotic POTs share an overall conserved binding site, individual amino acids changes in or in close vicinity of the binding site are likely responsible for observed differences in affinities and selectivity for particular peptides and drugs among the studied POT homologues.…”