Extracellular domain of PepT1 interacts with TM1 to facilitate substrate transport

Abstract

Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of small peptides and are essential for their absorption. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has twelve transmembrane (TM) helices that fold into an N-terminal domain (NTD, TM1-6) and a C-terminal domain (CTD, TM7-12) and has a large extracellular domain (ECD) between TM9-10. It is well recognized that peptide transport requires movements of the NTD and CTD, but the role of the ECD in PepT1 remains unclear. Here we report the structure of horse PepT1 encircled in lipid nanodiscs and captured in the inward-open apo conformation. The structure shows that the ECD bridges the NTD and CTD by interacting with TM1. Deletion of ECD or mutations to the ECD-TM1 interface impairs the transport activity. These results demonstrate an important role of ECD in PepT1 and enhance our understanding of the transport mechanism in PepT1.

Keywords: ECD; PepT1; SLC15; cryo-EM; nanodisc; transporter.

Figure 1.. Functional characterization of horse PepT1 with cell-based assays.

a, Peptide uptake activity of horse PepT1 WT and ΔECD in HEK293 cells. Error bars represent standard error of the mean (SEM) from three repeats. b, pH changes induced by proton-coupled peptide uptake. Ligand Ala-Ala (red) or blank buffer (dark gray) was added to HEK293 cells expressing horse PepT1 WT. Cells transfected with empty vector were added with the same ligand (light gray). The fluorescence changes (F/F₀) of a pH-sensitive dye were used to indicate cytosolic pH changes. The time courses are shown as solid line (mean) with shaded region (standard deviation, SD) from four repeats. c, Intracellular pH changes after 10 min of transport in the outside buffer of different pHs. Two-way ANOVA: among different pHs, p < 0.0001; with or without ligand, p < 0.0001; interaction, p < 0.0001. For all bar graphs in this paper, a scatter plot is overlaid on each bar and the height is the mean of at least three repeats with the error bar representing the SEM.

Figure 2.. Cryo-EM structure and ligand binding sites of horse PepT1.

a, SEC profile and SDS-PAGE gel image (inset) show the reconstitution of horse PepT1 into MSP1D1 nanodisc (solid blue line). Fractions within the blue dotted lines were collected for Cryo-EM grids. The SEC profile of horse PepT1 in DDM (black dash line) is shown for the comparison of its elution volume to that of a dimeric (~75 kDa per protomer, UniProt ID: Q9FY46) and monomeric transporter (~63 kDa, UniProt ID: A0A1U7U6F1) marked with red and orange ticks, respectively, on X-axis. b, Cryo-EM map of horse PepT1 in nanodisc (contoured at 6σ) colored as described in c. A gaussian smoothened map (translucent gray) is overlaid to display the contour of nanodisc density around the transporter. c, Structure of horse PepT1 in cartoon representation shows the inward-open conformation with NTD in orange, intracellular domain helix (ICH) bundle in red, and CTD in blue. d, Structural alignment of horse PepT1 with human PepT2 bound with an Ala-Phe ligand (PDB ID: 7PMY) colored in gray. Conserved charged residues in the ligand binding pocket are shown as stick. e, Cut-away view of the electrostatic surface in the binding pocket of horse PepT1 show the opposite charges on the NTD (positive) and CTD (negative). The ligand Ala-Phe shown in sphere are superimposed. f, Functional characterization of mutations in the ligand binding pocket. The Dunnett’s test was used as a post hoc test following one-way analysis of variance (ANOVA) with the WT group as control. Statistical significances are indicated: ****, p < 0.0001.

Figure 3.. Interactions between the ECD and NTD in horse PepT1.

a, The structure of horse PepT1 shows interactions between ECD and NTD mediated by K483 and TM1. E482 and K483 from NTD and residues on the extracellular side of TM1 and TM2 are shown as stick with surrounding electron density shown as translucent gray surface at 4σ. Distance measurements are indicated as yellow dash lines with labeled distances in Å. b, The structure of human PepT2 (PDB ID: 7PMY) shows that H76 cap the C-terminus of TM1. The electron density around H76 is shown at 6.5σ. Transport activities of horse PepT1 with mutations that affect the ECD-NTD interactions measured in the cell-based proton transport assay (c) and peptide uptake assay (d). The Dunnett’s test was used as a post hoc test following one-way ANOVA with the WT group as control. Statistical significances are indicated: *, p < 0.05; **, p < 0.01; ****, p < 0.0001.