Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2

Abstract

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.

Figure 1

Structure of PepT_So. (A) PepT_So topology. The central and extracellular cavities are shown as a closed diamond and open triangle, respectively. A bound ligand in the central cavity is represented as a black horizontal bar. Functionally important residues conserved between PepT_So and metazoan peptide transporters are highlighted by shapes in Supplementary Figure S1 and mapped onto the topology diagram. (B) PepT_So structure viewed in the plane of the membrane. The two hydrophilic cavities present in the structure are outlined in dashed lines. The hydrophobic core of the membrane (pale yellow) is distinguished from the interfacial region (light grey). N and C represent the N- and C-termini, respectively. Bound ligand is shown in black. Helices are labelled. (C) View from the extracellular side of the membrane.

Figure 2

Transport of peptides by PepT_So. (A). Concentration dependence of PepT_So-mediated glycylsarcosine (Gly-Sar) uptake in E. coli. Results shown, expressed per milligram of His-tagged PepT_So protein, are mean values±s.d. (n=4). (B) Effect on transport activity of mutating His61 to cysteine. Uptake of [³H]-glycylsarcosine (Gly-Sar) over a period of 10 min was measured in E. coli cells expressing the indicated forms of His-tagged PepT_So or in control cells lacking the transporter. Results shown are mean values±s.d. (n=3) and are expressed per milligram dry weight of bacteria. The inset shows western blots of equivalent samples from each culture, stained with a monoclonal antibody against oligohistidine. (C) Extracellular cavity viewed in the membrane plane. The central and extracellular cavities are isolated from each other by a putative extracellular gate. Residues in the central and extracellular cavities are highlighted in red and yellow, respectively. His61, part of the proposed proton–substrate coupling machinery is shown in green. Bound ligand is shown as a black CPK model of a di-alanine peptide. (D) Intracellular gate viewed in the membrane plane. Residues forming the gate are shown as stick models with transparent CPK surfaces. LacY helices (grey) are superposed onto PepT_So. Bound ligand is shown as a black CPK model as in C.

Figure 3

Comparison of PepT_so and LacY structures. Electrostatic surface representation showing the location of the hydrophilic cavities in a section through the protein volumes of (A) PepT_So and (B) LacY. The N- and C-terminal six-helix bundles are labelled. (C) Superposed transmembrane helices of PepT_So and LacY viewed from the intracellular side of the membrane. PepT_So helices are labelled and shown in yellow except for helix H2 (green) and helices H7, H11 and H12 (red), which form sub-bundle C1. The N-terminal six-helix bundle and the C-terminal sub-bundles C1 and C2 are highlighted. LacY helices are shown in cyan. Bound ligand is shown as a black CPK model of a di-alanine peptide. Helices HA and HB have been omitted for clarity.

Figure 4

The peptide-binding site. Stereo view of the central cavity as viewed from above on the extracellular side of the membrane. Conserved residues between PepT_So and the mammalian peptide transporters are labelled and coloured according to side-chain type, Arg and Lys (blue), Glu and Ser (red), Tyr (green) and Trp, Phe and Leu (cyan). A di-peptide sized C_α baton (orange) is fitted as a size reference into the mFo-DFc electron density observed in the central cavity (blue mesh), contoured at 4 σ.

Figure 5

A possible mechanism for peptide–proton symport. (A) Outward-facing state: peptide (Pep) and proton (H⁺) can access respective binding sites through the outward-facing cavity that is open towards the extracellular side of the membrane. The peptide-binding site is made from the surfaces of both the N- and C-terminal helix bundles (indicated by + and − signs), whereas the proton-binding site is located in the area close to the extracellular gate. (B) Occluded state: both ends of the central cavity are closed with peptide occluded into the central cavity. The proton-binding site is still exposed to the extracellular side through the extracellular cavity. (C) Inward-facing state: peptide and proton are released on the intracellular side of the membrane through the inward-facing cavity. Note that the proton-binding site is exposed to the intracellular side in this conformation.