Molecular insights into proton coupled peptide transport in the PTR family of oligopeptide transporters

Abstract

Background: Cellular uptake of small peptides is an important physiological process mediated by the PTR family of proton-coupled peptide transporters. In bacteria peptides can be used as a source of amino acids and nitrogen. Similarly in humans peptide transport is the principle route for the uptake and retention of dietary protein in the form of short di- and tri-peptides for cellular metabolism.

Scope of the review: Recent crystal structures of bacterial PTR family transporters, combined with biochemical studies of transport have revealed key molecular details underpinning ligand promiscuity and the mechanism of proton-coupled transport within the family.

Major conclusions: Pairs of salt bridge interactions between transmembrane helices work in tandem to orchestrate alternating access transport within the PTR family. Key roles for residues conserved between bacterial and eukaryotic homologues suggest a conserved mechanism of peptide recognition and transport that in some cases has been subtly modified in individual species.

General significance: Physiological studies on PepT1 and PepT2, the mammalian members of this family, have identified these transporters as being responsible for the uptake of many pharmaceutically important drug molecules, including antibiotics and antiviral medications and demonstrated their promiscuity can be used for improving the oral bioavailability of poorly absorbed compounds. The insights gained from recent structural studies combined with previous physiological and biochemical analyses are rapidly advancing our understanding of this medically important transporter superfamily. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

Keywords: Drug transport; Major facilitator superfamily; PTR/NRT1/POT family; Peptide transport.

Graphical abstract

Fig. 1

A. Sequence alignment and secondary structure of PepT_So. Amino acid sequence alignment of S. oneidensis PepT_So (Uniprot: Q8EKT7), S. thermophilus PepT_St (Q5M4H8), G. kaustophilus GkPOT (Q5KYD1), S. oneidensis PepT_So2 (QHE8ES) with human PepT1 (B2CQT6) and PepT2 (Q16348) homologues using ClustalW. Identical residues are highlighted in red. The α-helices in PepT_So are depicted as coils above the sequences. The conserved signature motifs within the PTR family are marked with orange horizontal bars. Residues described in the review are highlighted; proton binding (green triangles), peptide transport (yellow ovals), peptide specificity (gold stars) and extracellular and intracellular gate (blue squares). The horizontal blue bar represents the location of the extracellular domain present in the mammalian homologues but absent in the prokaryotic members. B. Topology diagram of human PepT1 with helices colored blue to red including the position of the extracellular ecto domain between TM9 and 10. The locations of the conserved sequence motifs within the TM helices are shown. Sequence logos (http://weblogo.berkeley.edu/) of these motifs generated from 24 eukaryotic and 19 prokaryotic PTR family sequences aligned as described for human PepT1 and PepT2 are illustrated.

Fig. 2

Crystal structures of PTR family transporters. Four crystal structures of PTR/POT family transporters have been determined to date. These represent three unique states in the alternating access transport cycle; ligand bound occluded (PepT_So), ligand bound inward open (PepT_So2 & GkPOT) and ligand free inward open (PepT_St & GkPOT), shown here in ribbon representation colored from their N-terminus (blue) to C-terminus in their respective states in a simplified model of the alternating access transport cycle. The PDB codes for each structure are given in parentheses.

Fig. 3

Structure of GkPOT^E310Q bound to the phosphonopeptide alafosfalin. A. View through a section of the protein volume in the plane of the membrane showing the central peptide binding site (dashed box) and alafosfalin peptide in sticks. B. Close up view of the binding site rotated 90° to the view in A. Hydrogen bonds between the binding site residues and alafosfalin peptide are shown as magenta dashed lines. The chemical structure of alafosfalin illustrates the similarities and differences to naturally occurring peptides.

Fig. 4

Structure of PepT_So2 bound to the phosphonopeptide alafosfalin. A. View through a section of the protein volume in the plane of the membrane showing the central peptide binding site (dashed box) and alafosfalin peptide in sticks. B. Close up view of the binding site rotated 90° to the view in A. Hydrogen bonds between the binding site residues and alafosfalin peptide are shown as magenta dashed lines.

Fig. 5

Structural comparison of the alafosfalin binding site between GkPOT^E310Q and PepT_So2. A. Binding site of both GkPOT (colored residues) and PepT_So2 (gray residues) is shown. The areas that differ most are highlighted by the dashed magenta ovals. B. View rotated 90° with TM8 removed for clarity. The black arrow indicates the displacement of the alafosfalin peptide (~ 2 Å) in GkPOT relative to that in PepT_So2.

Fig. 6

Peptide binding site. A. Zoomed in view of the central cavity in PepT_St, with the helices represented as cylinders and shown in the plane of the membrane. Side chains observed within the cavity are labeled, with the equivalent residue numbers for GkPOT shown in parenthesis. The function of these residues determined from in vitro assays is indicated. B. Table showing the calculated IC₅₀ values for different peptides in the WT and Y29F and Y68F variants of PepT_St.

Fig. 7

Structural comparison between PepT_So and PepT_St revealed the nature of the extracellular and intracellular gates. Centre of the figure shows a slice through the volume of PepT_So and PepT_St positioned as in Fig. 3 & 4 for GkPOT and PepT_So2 respectively. Key structural regions discussed in the text are illustrated. Top left, zoomed in view of the extracellular gate region in PepT_So, illustrating the salt bridge interaction between Arg32 on TM1 and D316 on TM7 and their position with respect to the extracellular cavity. Top right, zoomed in view of the equivalent region in PepT_St, showing the proximal and distal salt bridges stabilizing the interaction between helices TM1, TM2 from the N-terminal and TM7, TM8 from the C-terminal bundles respectively.

Fig. 8

Intracellular gate mechanism. A. Comparison between the inward open PepT_St (gray helices) and occluded PepT_So structure (colored helices) with arrows showing the hinge like movement that opens the intracellular gate. View is from the membrane plane. Residues forming the intracellular gate are shown as stick models with transparent CPK surfaces. Residue numbers are for PepT_St. The peptide-binding site containing Lys127 and Glu419 is indicated and alafosfalin modeled in the position occupied in the GkPOT structure. TM11 of PepTSo2 is also shown (dark gray) although in this case the movement of TM11 was more pronounced. B. The occlusion of peptide in the binding site appears to form via an induced fit mechanism with TM4–5 and TM10–11 closing in around the peptide (blue arrows). The four TM helices forming the intracellular gate are colored red. This movement is facilitated by conserved proline residues identified in the MD simulations as being important for the closing of TM4–5 in GkPOT and a conserved glycine and tryptophan residue in TM10 and TM11 respectively that facilitate the reciprocal movement in these helices illustrated in A. The alafosfalin peptide is shown in sticks.

Fig. 9

A model for proton driven peptide symport in the PTR family. A. An outward facing state, here modeled on the outward facing fucose permease structure (PDB: 3O7Q) is arbitrarily chosen at the start of the cycle. This state is characterized by the packing of helices TM4–5 with TM10–11 that form the intracellular gate and is potentially stabilized through a salt bridge interaction between K136 and E413 (GkPOT numbering). B. Peptide (here illustrated by the alafosfalin peptide in magenta sticks) and proton H⁺ (red circle) bind from the extracellular side of the membrane. The conserved glutamate on TM7 (E310) is where present likely to play an important role in proton binding, which must facilitate entry of the peptide. Additional important roles in proton binding for the N-terminal ExxERFxYY motif on TM1 and K136 in TM4 are also suggested by the functional data on PepTSt. Conserved tyrosine residues in the binding site play important roles in peptide recognition and specificity. C. Binding results in closure of the extracellular gate to form the occluded state, here modeled on the occluded structure. This conformation is characterized by the packing of helices TM7–8 against TM1–2 at the extracellular side of the binding site, assisted through the formation of the salt bridge interactions between R43 and E310 and by the distal salt bridge between TM2 and TM7 (not shown). Binding of both peptide and proton is also likely to disrupt the proposed interaction between K136 and E413, thereby facilitating release of the intracellular gate. In the occluded state an additional extracellular cavity may also form in the C-terminal domain following closure of the extracellular gate, as observed in the occluded PepT_So structure. D. Transition to the inward facing state occurs in part through localized hinge-like movement in helices TM4–5 and TM10–11 that results in release of the intracellular gate, allowing exit of proton and peptide into the interior of the cell. The closing of the extracellular gate and the formation of the salt bridge between R43 and E310 is very likely to result in release of the proton from E310 and this is likely to be coupled to the conformation changes that result in the opening of the intracellular gate helices and ejection of the peptide and proton into the interior of the cell.