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

Abstract

Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The E. coli genome codes for four different POTs, known as Di- and tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa Pro-macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 Å. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.

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.…”

“…Transport inhibition experiments indicated that the binding sites of DtpA and DtpB interact with a large number of substrates and peptidomimetic drugs in a similar fashion as the extensively studied mammalian homolog PepT1 16,[19][20][21][22][23][24] . DtpC and DtpD, however, were classified as atypical POTs, as they were shown previously to favour positively charged peptides as substrates 18,22,[25][26][27][28][29][30] .…”

“…In E. coli , four members of the POT family were identified and termed di- and tripeptide permease (Dtp) A to D 15–17 . Experimental structures of DtpA, DtpC and DtpD were reported recently 7,8,18 . Transport inhibition experiments indicated that the binding sites of DtpA and DtpB interact with a large number of substrates and peptidomimetic drugs in a similar fashion as the extensively studied mammalian homolog PepT1 16,19–24 .…”

“…In E. coli, four members of the POT family were identified and termed di-and tripeptide permease (Dtp) A to D [15][16][17] . Experimental structures of DtpA, DtpC and DtpD were reported recently 7,8,18 .…”

“…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).…”