Structural basis for polyspecificity in the POT family of proton-coupled oligopeptide transporters

Abstract

An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochemically distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.

Keywords: POT/PTR family; crystallography; major facilitator superfamily; membrane protein; peptide binding site.

Figure 1. Structure of PepT_St with Ala-Phe dipeptide

A Side on view of PepT_St with the 7.8 MAG lipid shown in spheres. The mF_o-DF_c difference electron density map (green) is shown, contoured at 3.0 σ. B Extracellular view of the peptide-binding site with hydrogen bonds as dashed lines. C Flexibility in the C-terminal domain indicated by the atomic displacement-coded putty thickness and colour gradient from blue (low disorder) to red (high disorder). The average atomic displacement parameter for the protein is 39.7 Ų. D View into the conserved hydrophobic pocket accommodating the ligand’s phenylalanine side chain in the peptide.

Figure 2. Tri-alanine binds to PepT_St in a vertical orientation

A View of the binding site in the plane of the membrane, showing the mF_o-DF_c difference electron density map, contoured at 3.0 σ. Hydrogen bonds are shown as dashed lines. In this panel, the peptide was modelled with the C-terminus orientated towards the apex of the cavity. B Electrostatic surface representation of the binding site in the Ala-Phe complex structure with both peptides superimposed illustrating the relative orientations. The side chains of key residues involved in peptide binding are outlined. C Effect of binding site mutations on proton-driven di-alanine uptake in liposomes. D IC₅₀ competition curves for di-alanine. E IC₅₀ competition curves for tri-alanine showing residual uptake of [³H]-di-Ala peptide normalized to WT. Data information: Error bars indicate the standard deviation from three independent experiments.

Figure 3. Conformational changes in PepT_St upon Ala-Phe binding

A Three states of PepT_St are shown superimposed; Apo (grey), tri-Ala bound (purple) and Ala-Phe bound (coloured from the N-terminus blue to C-terminus red). The arrows indicate the major structural change observed between the crystal structures, and black dots identify the main hinge points in the N- and C-terminal bundles, respectively. B Structural comparison between the three structures reveals that in the Ala-Phe complex helices H5, H8 and H11 close in around the peptide. The side chains of two conserved asparagines make hydrogen bonds to the Ala-Phe peptide at the hinge points in the helices H5 (N156) and H8 (N328). C Zoomed in view of H11 showing the formation of the hydrophobic pocket around the phenylalanine side chain of the Ala-Phe peptide (coloured helices) and the dissolution of the pocket upon adopting the more open structure observed in the Apo structure (grey).

Figure 4. A multi-site model for peptide transport in the POT family

In the outward-open state, the transporter will accept peptides and accommodate them in preferred orientations. This study has revealed two modes of binding, a lateral mode observed for the Ala-Phe peptide and a vertical mode for the tri-Ala peptide. In both cases, peptide and proton binding will drive the transporter to the occluded state, where the extracellular gate (H1-H7) is closed, stabilized by a salt bridge between Arg33 and Glu300 and the intracellular gate (H4-H10) salt bridge between Lys126 and Glu400 is disrupted . The structural transition required to drive reorientation of the binding site is potentially the result of proton binding to/release from Glu300 . Peptide release from the transporter will occur following rearrangement of the binding site to the inward-open state characterized by a disrupted hydrophobic pocket and concomitant proton release into the cytoplasm.