Structural snapshots of human PepT1 and PepT2 reveal mechanistic insights into substrate and drug transport across epithelial membranes

Abstract

The uptake of peptides in mammals plays a crucial role in nutrition and inflammatory diseases. This process is mediated by promiscuous transporters of the solute carrier family 15, which form part of the major facilitator superfamily. Besides the uptake of short peptides, peptide transporter 1 (PepT1) is a highly abundant drug transporter in the intestine and represents a major route for oral drug delivery. PepT2 also allows renal drug reabsorption from ultrafiltration and brain-to-blood efflux of neurotoxic compounds. Here, we present cryogenic electron microscopy (cryo-EM) structures of human PepT1 and PepT2 captured in four different states throughout the transport cycle. The structures reveal the architecture of human peptide transporters and provide mechanistic insights into substrate recognition and conformational transitions during transport. This may support future drug design efforts to increase the bioavailability of different drugs in the human body.

Fig. 1.. Cryo-EM structures of apo HsPepT1 and HsPepT2 bound to Ala-Phe.

(A) Whole-cell transport competition assays of the β-Ala-Lys peptide coupled to the fluorescent AMCA moiety (AK-AMCA) in HsPepT2-transfected HEK293 cells showing reduced AK-AMCA uptake in the presence of 5 mM of the competing substrate. (B and C) Three-dimensional reconstructions of (B) HsPepT1 and (C) HsPepT2 with corresponding 2D class averages and surface representation highlighting the (B) outward-facing open and (C) inward-facing partially occluded conformations.

Fig. 2.. Overall architecture of human POTs.

(A) Apo-HsPepT1 and (B) substrate-bound HsPepT2 models shown as cartoon representation. The different architectural elements are labeled. Loops that could not be modeled because of poor density are shown as dashed lines.

Fig. 3.. Structural comparison between the outward- and inward-facing states observed in apo HsPepT1 and substrate-bound HsPepT2.

(A) Opening and closing of the substrate binding site to the extracellular and intracellular milieu observed in HsPepT1 (blue) and HsPepT2 (green). (B) The distances between C_α atoms of the relevant pairs of helix tips from all bacterial POTs determined by x-ray crystallography were measured and compared to the human transporters. (C) Rocking motions of the N-bundle (HsPepT1: light blue and HsPepT2: light green) and C-bundle (HsPepT1: dark blue and HsPepT2: dark green) after structural alignment of both transporter units. (D) Bending of TMs with measured tilt angles observed in the N-bundle (left) and C-bundle (right) between HsPepT1 and HsPepT2.

Fig. 4.. Interactions stabilizing the outward-facing open state of HsPepT1 and the inward-facing partially occluded state of HsPepT2.

The locations of key interactions are shown and labelled on (A) HsPepT1 and (B) HsPepT2. Corresponding close-up views show the cryo-EM densities of the side chains forming the interactions as indicated by dashed lines.

Fig. 5.. Structural basis for substrate recognition in human POTs.

(A) Concentration-dependent competition assay of the β-Ala-Lys peptide coupled to the fluorescent AMCA moiety (AK-AMCA) in HsPepT2 with the dipeptide Ala-Phe. The average uptake value for each condition was calculated from three independent measurements. The error bars correspond to the SD from these independent measurements. (B) Thermal stabilization of detergent-solubilized HsPepT2 upon substrate binding measured by nano-differential scanning fluorimetry (DSF) at increasing concentrations of Ala-Phe (inset shows the increase in melting temperature with increasing peptide concentration). (C) Close-up view of the HsPepT2 peptide binding site. Electrostatic interactions between the peptide (shown in orange) and the transporter (shown in green) are displayed as gray dashes. The interaction between Y94 and the C-ter pocket is shown as green dashes. The different pockets are indicated. (D) Thermal stabilization of detergent solubilized HsPepT1 upon substrate binding measured by nano-DSF at increasing concentrations of Ala-Phe. (E) Three-dimensional reconstruction of outward-facing open HsPepT1 bound to Ala-Phe (F) Overlay of the binding sites of outward-facing open apo (shown in gray) and Ala-Phe–bound HsPepT1 (shown in blue colors). Electrostatic interactions between the peptide (shown in orange) and the transporter are displayed as gray dashes. The interaction between Y64 and the C-ter pocket is shown as blue dashes.

Fig. 6.. Mechanism for substrate recognition and transport in human POTs based on the presented structures.

(A) In the first step of the transport cycle, the transporter is in an outward-facing open state stabilized by two salt bridges between R159-E604 and R161-D341. (B) Upon peptide binding—accommodated in the charged central cavity via its N terminus by N171, N329, and E595 and optionally its C terminus by R27 and K140—the N-bundle helices follow bending and rigid body motions resulting in tightening of the central cavity. (C) Further bending of TM2 allows the interaction of H57, S302, N630, and D298 as a crucial step before (D) total sealing of the extracellular side and switching to the inward-facing occluded state stabilized by the salt bridge R185-D323. (E) Last, opening of the cytosolic side is achieved by TM4 and TM5 moving away from TM10 and TM11 resulting in the loss of the crucial interaction between the transporter and the peptide termini, allowing substrate release to the cytoplasm. The structures shown in (A), (B), and (C), represent models of HsPepT1 on the basis of the three different cryo-EM maps presented in this article. Missing loops have been added. The structure in (D) is derived from the experimental HsPepT2 structure. (E) corresponds to the AlphaFold structure prediction (50) available in the EMBL-EBI AlphaFold database. Numbering of residues illustrated in this model follow the HsPepT1 nomenclature. Conformational changes along the reaction cycle are colored according to the root mean square deviation (RMSD) between different state structures.