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

2010; Springer Nature; Volume: 30; Issue: 2 Linguagem: Inglês

10.1038/emboj.2010.309

1460-2075

Simon Newstead, David Drew, Alexander D. Cameron, Vincent L. G. Postis, Xiaobing Xia, P. W. Fowler, Jean C. Ingram, Elisabeth P. Carpenter, Mark S.P. Sansom, Michael J. McPherson, Stephen A. Baldwin, So Iwata,

DNA and Nucleic Acid Chemistry

Article3 December 2010free access Crystal structure of a prokaryotic homologue of the mammalian oligopeptide–proton symporters, PepT1 and PepT2 Simon Newstead Corresponding Author Simon Newstead Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UK Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, JapanPresent address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK Search for more papers by this author David Drew David Drew Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, Japan Search for more papers by this author Alexander D Cameron Alexander D Cameron Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UK Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, Japan Search for more papers by this author Vincent L G Postis Vincent L G Postis Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Search for more papers by this author Xiaobing Xia Xiaobing Xia Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UKPresent address: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK Search for more papers by this author Philip W Fowler Philip W Fowler Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Jean C Ingram Jean C Ingram Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Search for more papers by this author Elisabeth P Carpenter Elisabeth P Carpenter Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UKPresent address: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK Search for more papers by this author Mark S P Sansom Mark S P Sansom Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Michael J McPherson Michael J McPherson Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Search for more papers by this author Stephen A Baldwin Corresponding Author Stephen A Baldwin Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Search for more papers by this author So Iwata Corresponding Author So Iwata Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UK Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, Japan Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Systems and Structural Biology Centre, RIKEN, Yokohama, Japan Search for more papers by this author Simon Newstead Corresponding Author Simon Newstead Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UK Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, JapanPresent address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK Search for more papers by this author David Drew David Drew Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, Japan Search for more papers by this author Alexander D Cameron Alexander D Cameron Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UK Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, Japan Search for more papers by this author Vincent L G Postis Vincent L G Postis Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Search for more papers by this author Xiaobing Xia Xiaobing Xia Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UKPresent address: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK Search for more papers by this author Philip W Fowler Philip W Fowler Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Jean C Ingram Jean C Ingram Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Search for more papers by this author Elisabeth P Carpenter Elisabeth P Carpenter Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UKPresent address: Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, UK Search for more papers by this author Mark S P Sansom Mark S P Sansom Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Michael J McPherson Michael J McPherson Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Search for more papers by this author Stephen A Baldwin Corresponding Author Stephen A Baldwin Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK Search for more papers by this author So Iwata Corresponding Author So Iwata Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UK Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, Japan Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Systems and Structural Biology Centre, RIKEN, Yokohama, Japan Search for more papers by this author Author Information Simon Newstead 1,2,3, David Drew1,3, Alexander D Cameron1,2,3, Vincent L G Postis4, Xiaobing Xia4, Philip W Fowler5, Jean C Ingram4, Elisabeth P Carpenter1,2, Mark S P Sansom5, Michael J McPherson4, Stephen A Baldwin 4 and So Iwata 1,2,3,6,7 1Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, UK 2Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UK 3Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Kyoto, Japan 4Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK 5Department of Biochemistry, University of Oxford, Oxford, UK 6Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan 7Systems and Structural Biology Centre, RIKEN, Yokohama, Japan *Corresponding authors: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: +44 1865 613 319; Fax: +44 1865 613 201; E-mail: [email protected] Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. Tel.: +44 1133 433 173; Fax: +44 1133 433 167; E-mail: [email protected] of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London, SW1 2AZ, UK. Tel.: +44 2075 941 873; Fax: +44 2075 943 022; E-mail: [email protected] The EMBO Journal (2011)30:417-426https://doi.org/10.1038/emboj.2010.309 There is a Have You Seen? (July 2012) linked with this article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 PepTSo, 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 PepTSo represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2. Introduction The absorption of dietary nitrogen in the form of peptides by plasma membrane transporters belonging to the solute carrier (SLC) 15 family is essential for human health. Evolutionarily, these transporters form part of the widely distributed proton-dependent oligopeptide transporter (POT) family (TC 2.A.17), also referred to as the peptide transporter or PTR2 family (Paulsen and Skurray, 1994; Steiner et al, 1995), members of which transport peptides, amino acids and nitrate (Huang et al, 1999). They are proton-driven symporters, and in both eukaryotes and prokaryotes use the inwardly directed proton (H+) electrochemical gradient to drive the uptake of peptides across cell membranes (Ganapathy and Leibach, 1983; Daniel et al, 2006). Human PepT1 (SLC15A1) is found predominantly in the small intestine, whereas PepT2 (SLC15A2) is found in the kidney, the lungs and central nervous system (Daniel and Kottra, 2004). PepT1 and PepT2 are predicted to contain 12 transmembrane (TM) helices with both N- and C-termini facing the cytoplasm, as is typical for major facilitator superfamily (MFS) members (Fei et al, 1994; Covitz et al, 1998). PepT1 is a high-capacity, low-affinity transporter and is the main route for dietary peptide uptake, whereas PepT2 operates as a low-capacity, high-affinity transporter, thought to mediate more selective transport in the kidney and other tissues (Terada et al, 1997; Doring et al, 2002; Daniel and Kottra, 2004; Biegel et al, 2006). In addition to peptides, the human proteins transport a broad spectrum of orally administered drugs, including the β-lactam antibiotics (Wenzel et al, 1995; Tamai et al, 1997; Faria et al, 2004), and are under active clinical investigation to improve the pharmacokinetic properties of antivirals such as valacyclovir (Ganapathy et al, 1998) and the vasopressor midodrine (Tsuda et al, 2006). To study the function of the mammalian transporters, a number of distantly related prokaryotic homologues with similar substrate specificities have been employed as model systems (Hagting et al, 1994; Harder et al, 2008; Ernst et al, 2009). Here, we report the crystal structure of a peptide transporter from the bacterium Shewanella oneidensis, PepTSo, which shows a high degree of sequence conservation within the TM region (∼30% identity) to the mammalian PepT1 and PepT2 proteins. All previously identified residues proposed to be functionally important in the mammalian transporters are conserved, including a critical histidine residue (Uchiyama et al, 2003) (His57 in human PepT1) (Supplementary Figure S1). The structure of PepTSo reveals important information concerning the spatial arrangement of residues involved in peptide and drug transport as catalysed by mammalian peptide transporters. In addition, it represents a ligand-bound occluded conformation for an MFS symporter, providing fresh insight into the alternating access model of membrane transport. Results Structure of PepTSo PepTSo contains 14 TM helices (Figure 1A), of which helices H1–H12 adopt the overall fold observed previously for the MFS transporters LacY, GlpT and EmrD (Figure 1B) (Abramson et al, 2003; Huang et al, 2003; Yin et al, 2006). The arrangement of the helices is also consistent with the EM projection structure of a previously studied POT protein, DtpD from Escherichia coli (Casagrande et al, 2009). Like previous MFS transporter structures, the N- and C-terminal six-helix bundles, formed by helices H1–H6 and H7–H12, come together to form a ‘V’-shaped transporter, related by a pseudo two-fold symmetry axis running perpendicular to the membrane plane. PepTSo has two additional TM helices, HA and HB, which are inserted into the cytoplasmic loop connecting the N- and C-terminal bundles. These form a hairpin-like structure in the membrane that packs against the periphery of the protein (Figure 1C). Their role is currently unclear. The apparent absence of these helices in the fungal, plant and metazoan protein sequences, however, suggests they do not contribute to any conserved transport mechanism. The position of PepTSo within the membrane has been examined using coarse-grained lipid bilayer self-assembly simulations (Scott et al, 2008). These demonstrate that PepTSo, including the hairpin helices HA and HB, reproducibly inserts into a modelled bilayer (Supplementary Figure S2). Figure 1.Structure of PepTSo. (A) PepTSo 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 PepTSo and metazoan peptide transporters are highlighted by shapes in Supplementary Figure S1 and mapped onto the topology diagram. (B) PepTSo 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. Download figure Download PowerPoint The apparent KM for transport of the hydrolysis resistant di-peptide glycylsarcosine is 1.5±0.15 mM, similar to the value reported for human PepT1 of 1.1±0.1 mM (Brandsch et al, 1994) (Figure 2A). Uptake of a fluorescent di-peptide, β-Ala-Lys-Ne-7-amino-4-methylcoumarin-3-acetic acid (β-Ala-Lys-(AMCA)) in cells overexpressing the PepTSo gene was reduced upon addition of either di- or tri-alanine peptides to the media (Supplementary Figure S3). Addition of L-alanine or the larger tetra-alanine peptide, however, had little effect, suggesting a similar preference for di- and tri-peptides as reported for the mammalian transporters (Fei et al, 1994). Uptake was also abolished by the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, consistent with a dependence on the proton electrochemical gradient (ΔμH+) to drive transport. The crystal structure was solved by multiple isomorphous replacement with anomalous scattering using mercury derivative crystals and seleno-L-methionine incorporated protein (Table I and Supplementary Tables I and II). Assignment of the amino-acid sequence to the density map was aided through identification of the 22 selenium and three mercury sites present in the molecule (Supplementary Figure S4). The model was built and refined using data with anisotropic truncation of the observed structure factors (Strong et al, 2006) to 4.3 Å along the A and B axes, while keeping the C axis at 3.6 Å (Supplementary Figure S5). The final model was refined to an Rfactor of 27.8% and a corresponding Rfree of 29.6% (Table I). There are three PepTSo molecules in the crystallographic asymmetric unit and their structures are identical in the context of this analysis. Figure 2.Transport of peptides by PepTSo. (A). Concentration dependence of PepTSo-mediated glycylsarcosine (Gly-Sar) uptake in E. coli. Results shown, expressed per milligram of His-tagged PepTSo 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 PepTSo 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 PepTSo. Bound ligand is shown as a black CPK model as in C. Download figure Download PowerPoint Table 1. Data collection and refinement statistics Native MMC-1a MMC-2 MMC-3 HgAc Se Space group P32 P32 P32 P32 P32 P32 a, b, c (Å) 159.4, 159.4, 153.0 159.7, 159.7, 153.9 157.6, 157.6, 153.1 158.1, 158.1, 153.6 159.7, 159.7, 153.9 157.6, 157.6, 153.1 α, β, γ (deg) 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 Resolution (Å) 40–3.6 (3.8–3.6)b 40–4.6 (4.8–4.6) 40–4.0 (4.1–4.0) 40–4.5 (4.7–4.5) 40–4.6 (4.8–4.6) 40–5.0 (5.2–5.0) Rmergec 8.9 (67.5) 9.3 (82.4) 10.4 (76.1) 16.0 (82.3) 9.4 (50.2) 10.9 (50.0) I/σI 9.0 (1.1) 7.1 (1.1) 8.8 (1.4) 7.7 (1.0) 8.5 (1.68) 7.5 (2.0) Completeness (%) 93.8 (88.7) 90.3 (90) 97.7 (96.4) 99.7 (99.0) 96.4 (95.7) 99.0 (99.0) Redundancy 2.0 (1.8) 1.7 (1.7) 3.2 (2.8) 3.3 (3.0) 2.4 (2.2) 3.3 (3.1) Resolution (Å) 19–3.6 (3.8–3.6) No of reflections 47 021 Rwork/Rfree 27.8 (35.8)/ 29.6 (40.4) No of protein atoms 10 533 R.m.s. deviations Bond lengths (Å) 0.009 Bond angles (deg) 1.12 For details on derivatisation see Supplementary Materials and methods. Values in parenthesis are for the highest-resolution shell. The last shell Rmerge is high for some of the derivative data because of severe anisotropy in the diffraction images. Hydrophilic cavities In the structure, we observe a central cavity and a smaller extracellular cavity, both of which are hydrophilic (Figure 1B). The central cavity is situated within the centre of the membrane and closed to the extracellular space by a gate made of helices H1, H2, H7 and H8, which pack closely together (Figures 1C and 2C). Previous secondary active transporter structures have all revealed a ligand-binding site located within the centre of the membrane, essentially equidistant between extracellular and intracellular sides (reviewed in Boudker and Verdon, 2010). Indeed, the residues extending into the central cavity in PepTSo are all known to affect peptide binding and/or transport in the mammalian proteins (Terada et al, 1996, 2004; Fei et al, 1997; Bolger et al, 1998; Yeung et al, 1998; Chen et al, 2000; Uchiyama et al, 2003; Hauser et al, 2005; Pieri et al, 2009; Xu et al, 2009). This cavity is therefore an obvious location for the peptide-binding site, as we discuss below. As further confirmation, we also observe clear electron density within this cavity for a bound ligand. The apparent Km for the substrate peptide glycylsarcosine is low, 1.5 mM; therefore, this density is unlikely to represent a co-purified natural peptide. The density is more likely to represent a non-natural ligand or a high-affinity inhibitor acquired from either the purification or the crystallization conditions. The position of this density corresponds to the same location of the bound sugar analogue β-D-galactopyranosyl-thio-β-D-galactopyranoside observed in the binding site of LacY (Abramson et al, 2003) (Supplementary Figure S6). Access to the cytoplasm from this cavity is restricted by an intracellular gate formed by side-chain interactions between two-helix hairpins, helices H4 and H5 on the N-terminal side, and H10 and H11 on the opposing C-terminal side (Figure 2D). The interaction between these helices occurs through residues that are conserved across the vertebrate peptide transporters (Figure 1A; Supplementary Figure S1). The most prominent of these interactions involves Leu427(603) on helix H10 packing against Tyr154(167) and Phe150(163) on helix H4, both of which form part of the highly conserved POT family PTR2_2 motif (FYxxINxG), suggesting a possible role in regulating the exit of peptides from the central cavity. Numbers in brackets correspond to the equivalent residues in human PepT1. Indeed, several point mutations within the PTR2_2 motif have been found to inactivate or greatly impair transport (Hauser et al, 2005). Considering the position of a bound ligand within the central cavity and its confinement through the closure of both extracellular and intracellular gates (Figure 3A and B), we have described the present state of PepTSo as substrate occluded, in analogy with the LeuT superfamily transporters (Krishnamurthy et al, 2009). Figure 3.Comparison of PepTso and LacY structures. Electrostatic surface representation showing the location of the hydrophilic cavities in a section through the protein volumes of (A) PepTSo and (B) LacY. The N- and C-terminal six-helix bundles are labelled. (C) Superposed transmembrane helices of PepTSo and LacY viewed from the intracellular side of the membrane. PepTSo 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. Download figure Download PowerPoint Comparing this conformation with previous MFS transporter structures has highlighted a potentially important structural feature of transport within the MFS. The various LacY structures are all in inward-facing open conformations (Abramson et al, 2003). The EmrD structure on the other hand likely corresponds to an occluded state, although no evidence of bound ligand could be observed in the electron density maps (Yin et al, 2006). Using a secondary structure matching algorithm (Krissinel and Henrick, 2004) to overlay these evolutionarily distinct proteins, it is clear that the structure of PepTSo is more similar to that of EmrD than to LacY, with an average distance of 2.4 Å for 155 equivalent Cα atoms (Supplementary Figure S7). This observation supports the conclusion that PepTSo also represents the occluded conformation for the POT family. To further understand the differences between PepTSo and LacY, which represent two different conformational states for MFS transporters, we also compared their structures by calculating the change in position between Cα atoms of related helical segments within the N- and C-terminal six-helix bundles, respectively (see Supplementary data). The main difference was clearly identified as residing within the C-terminal domain, the average distances between atoms that make up the N- and C-terminal helix bundles being 2.8 and 4.4 Å, respectively (Supplementary Table III). Further insight was drawn from comparing Cα displacements of individual helices within the C-terminal domain. In this case, helix H7 of PepTSo and LacY showed the largest average Cα displacement between the two structures of 5.7 Å. The C-terminal six-helix bundle of MFS transporters is made up of inverted repeats constructed from helices H7–H9 and H10–H12. Structurally, however, they also form two sub-bundles of helices consisting of H7, H11 and H12 (sub-bundle C1) and H8, H9 and H10 (sub-bundle C2) (Figure 3C). When the N-terminal domains of PepTSo and LacY are superimposed, the largest deviation is observed in sub-bundle C1, which is displaced by an approximate 11° rotation (Supplementary Figure S8). These helices seem to be the ones mainly responsible for the asymmetry between the N- and C-terminal helix bundles between the occluded conformation of PepTSo and inward open conformation of LacY. These observations suggest that a large conformational change takes place predominantly within the C-terminal domain of MFS transporters during the transition from ligand-bound occluded to inward open state. The observed extracellular cavity is also located at the interface between the N- and C-terminal domains and is roughly cone shaped, with the apex at the bottom near the central cavity, opening outward (Figures 1B and 2C). The overall dimensions of the cavity are ∼16 × 8 × 8 Å. Atomistic molecular dynamics (MD) simulations reproducibly show that both this extracellular cavity and the central cavity are fully solvated and not blocked by lipid molecules in the present conformation (Supplementary Figure S9). The EmrD structure exhibits a similar cavity (Supplementary Figure S7), which differs mainly in that its surface is composed primarily of hydrophobic residues. This observation supports the possibility that this cavity represents the vestiges of either an entrance (for PepTSo) or an exit (for EmrD) pathway for substrates, these being hydrophilic peptides for PepTSo and hydrophobic compounds for EmrD, when the central cavity is open towards the extracellular side. The peptide-binding site As previously noted, many of the residues conserved between PepTSo and the mammalian peptide transporters cluster around the central hydrophilic cavity, with approximate dimensions of 13 × 12 × 11 Å. These dimensions are sufficient to accommodate both di- and tri-peptides, although would be sterically restrictive for larger tetra-peptide ligands. This may explain the competition we observe in the in vivo transport assay between peptides of this size and the β-Ala-Lys-(AMCA) (Supplementary Figure S3). The dimensions of the cavity could also explain the lack of affinity for single amino acids, as these would presumably be incapable of interacting sufficiently with both the N- and C-terminal domains of the transporter. Sitting within the centre of the cavity we observe strong (>4σ) electron density for an unidentified ligand (Figure 4; Supplementary Figure S10) of approximately the same dimensions as a di-peptide. In the figure, a Cα model of a di-alanine peptide has been placed into the density as a reference to evaluate the size of the cavity, although no peptide was modelled during refinement. 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 PepTSo 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 Cα 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. Download figure Download PowerPoint The binding site is formed by residues from helices H1, H2, H4 and H5 from the N-terminal six-helix bundle and from helices H7, H8, H10 and H11 from the C-terminal bundle. On the N-terminal side of the binding site, three conserved positively charged residues, Arg25(27), Arg32(34) and Lys127(140) extend into the cavity. It has been reported that mutation of Arg25(27) in human PepT2 to a histidine completely inactivates transport (Terada et al, 2004). Two conserved tyrosine residues, Tyr29(31) and Tyr68(64), are positioned close to this positively charged cluster. On the C-terminal side of the binding site, at a distance of ∼13 Å from Lys127(140), are two further strictly conserved residues, Glu419(595) and Ser423(599), located in close proximity to Tyr154(167). Various mutants of Glu419(595) in PepT1 have been reported to drastically reduce transport activity, except where mutation was to an aspartic acid (Xu et al, 2009), indicating the importance of a negatively charged residue at this position. The arrangement of opposite charges within the binding site may have an important role in the recognition and orientation of peptides through the creation of a dipole moment. The presence of several possible hydrogen-bond donors and acceptors could be advantageous in adapting to peptides of various lengths, sequences and charges.