Cryo-EM structure of PepT2 reveals structural basis for proton-coupled peptide and prodrug transport in mammals

Abstract

The SLC15 family of proton-coupled solute carriers PepT1 and PepT2 play a central role in human physiology as the principal route for acquiring and retaining dietary nitrogen. A remarkable feature of the SLC15 family is their extreme substrate promiscuity, which has enabled the targeting of these transporters for the improvement of oral bioavailability for several prodrug molecules. Although recent structural and biochemical studies on bacterial homologs have identified conserved sites of proton and peptide binding, the mechanism of peptide capture and ligand promiscuity remains unclear for mammalian family members. Here, we present the cryo-electron microscopy structure of the outward open conformation of the rat peptide transporter PepT2 in complex with an inhibitory nanobody. Our structure, combined with molecular dynamics simulations and biochemical and cell-based assays, establishes a framework for understanding peptide and prodrug recognition within this pharmaceutically important transporter family.

Fig. 1. Functional characterization of PepT2 and the inhibitory nanobody D8.

(A) Location of PepT2 within the kidney (indicated by a box) and topology schematic of mammalian peptide transporters. (B) PepT2 reconstituted into liposomes can uptake peptide in the presence of a membrane potential generated through the presence of valinomycin. The schematic shows the experimental setup used. (C) PepT2 is a proton-driven peptide transporter being able to use a membrane potential (Δψ, negative inside) or a pH gradient to drive peptide accumulation. The combination of both gradients (ΔμH⁺) results in increased transport. (D) Most of nanobodies (NBs) raised against PepT2 lacking the extracellular domain (ECD) could inhibit peptide uptake. The NB referred to as D8 was the one used to solve the structure of PepT2. Inset: NB_D8 has a dissociation constant (K_d) of ~26 nM for binding to full-length PepT2; a representative trace from the biolayer interferometry is shown with the calculated mean and SD (n = 3).

Fig. 2. Cryo-EM structure of PepT2.

(A) Cryo-EM density of the PepT2 nanobody complex, contoured to a threshold level of 0.43. Left: The TM domain, ECD, and nanobody are represented in blue, green, and orange, respectively. Right: Rotated view of the TM domain, colored as rainbow. Inset: Superposition with lower contoured (threshold level of 0.13) volume to display the detergent micelle. (B) Cartoon representation of PepT2 highlighting the two domains, the membrane-embedded transport domain and the ECD composed of two IgG domains linked in tandem. (C) Cryo-EM density with overlaid model of ECD and nanobody. IgG-1 subdomain, IgG-2 subdomain, and nanobody are colored in green, violet, and orange, respectively. Map is contoured to a threshold level of 0.29.

Fig. 3. PCA of the dynamic movements of the ECD of PepT2.

(A) Decomposition of the ECD domain motion by PCA showing that most motion can be captured by the first four eigenvectors, shown in (B). Projections of the first four eigenvectors show that the motions are normally centered on the mean with no distinctive hotspots. (C) The first four motions can be classified as an x- or y-plane translation (purple and orange) and rotation along the z axis (green) and a z-axis translation (red), which can be summarized as translations in all three dimensions and a rotation of the IgG along the z axis with respect to the membrane. (D) Snapshot of the simulations showing PepT2 in a lipid bilayer and the role of the lateral helix in stabilizing the position of the transporter domain. The conserved arginine side chains on the lateral helix are shown (gray sticks).

Fig. 4. Outward open conformation of PepT2.

(A) Slice through the electrostatic surface representation of PepT2 as viewed in the plane of the membrane. The intracellular gating helices TM4 to TM5 and TM10 to TM11 are shown. (B) Zoomed-in view of the intracellular gate, with the key stabilizing interactions identified and labeled. (C) Cartoon representation showing the gating helices in PepT2. Key side chains involved in gate stabilization are shown. (D) Cell-based transport assays for PepT2 and variants of the intracellular gate–interacting side chains. (E) Western blot using an anti–green fluorescent protein (GFP) antibody to detect WT and variant forms of PepT2.

Fig. 5. Peptide binding to PepT2.

(A) Correlation between IC₅₀ values and ABFE [Spearman rank correlation: r_s = −0.9, P = 0.04; Pearson correlation coefficient, ABFE versus log(IC₅₀): r = −0.83, P = 0.08]. (B) Slice through the electrostatic surface representation of PepT2, with the peptides shown overlaid in the binding site. (C) Close-up view of the binding site shown in (B). The five peptides are shown as different colored sticks, with key side chains shown in sticks and labeled. Side-chain positions are taken from the cryo-EM structure. (D) Zoomed-in view of the binding site showing the different binding poses for l-Ala-Phe (magenta) and l-Phe-Ala (purple). (E) Schematic of the peptide binding site, highlighting the key structural features (see text) for Tyr-Tyr-Tyr recognition. The final binding pose for Tyr-Tyr-Tyr is shown at the inset, top right. Arrows indicate direction of hydrogen bonds, with ionic interactions depicted as straight lines.

Fig. 6. Binding of Ala-Ala-Ala proceeds via N-terminal engagement followed by C-terminal engagement.

(A) Free energy calculations show that N-terminal engagement followed by C-terminal engagement is more energetically favorable. (B) The N terminus of Ala-Ala-Ala initially interacts with Asp³¹⁷ before transferring to Glu⁶²² and is further coordinated by Asn¹⁹² and Asn³⁴⁸. (C). Once the N terminus has formed a stable interaction, the C terminus engages with Arg⁵⁷ from Lys⁶⁴ and the movement is smoothened by Tyr⁶⁰, Tyr⁶¹, and Tyr⁶⁴ (D).

Fig. 7. Transport mechanism of PepT2.

(A) Cartoon representation of PepT2 in the outward open state compared to PepT_So (PDB: 2XUT) in the inward open state. The N- and C-terminal six-helix bundles are marked with blue and red dotted lines, respectively. (B) Structural overlay of the N-terminal bundle (left) and C-terminal bundle (right) colored as in (A). The key structural changes in the N-terminal bundles are highlighted by yellow lines, showing the angles of movement in TM2, TM4, and TM11. Key side chains involved in peptide and proton binding are indicated (purple sticks) and labeled. The docked l-Ala-Phe peptide is shown for reference.

Fig. 8. Alternating access transport mechanism within the SLC15 family.

Schematic representation of the transport cycle for PepT2, detailed in the main text.

Fig. 9. Model for prodrug recognition by PepT2.

(A) Slice through the electrostatic surface representation of PepT2, showing the location of valacyclovir (PDB: TXC) docked into the peptide binding site. (B) Close-up view of the binding site shown in (A). (C) Structural overlay of the l-Phe-l-Ala-l-Gln peptide on the valacyclovir binding pose. (D) Representative IC₅₀ curves for valacyclovir and valganciclovir reporting calculated means and SD (n = 3). (E) Schematic of valacyclovir binding site, highlighting the key structural features in prodrug recognition. Arrows indicate direction of hydrogen bonds, with ionic interactions depicted as straight lines.