Alternating access mechanism in the POT family of oligopeptide transporters
2012; Springer Nature; Volume: 31; Issue: 16 Linguagem: Inglês
10.1038/emboj.2012.157
ISSN1460-2075
AutoresNicolae Solcan, Jane Kwok, P. W. Fowler, Alexander D. Cameron, David Drew, So Iwata, Simon Newstead,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoArticle1 June 2012Open Access Alternating access mechanism in the POT family of oligopeptide transporters Nicolae Solcan Nicolae Solcan Department of Biochemistry, University of Oxford, Oxford, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author Jane Kwok Jane Kwok Department of Biochemistry, University of Oxford, Oxford, 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 Alexander D Cameron Alexander D Cameron Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author David Drew David Drew Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author So Iwata So Iwata Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK Division of Molecular Biosciences, Imperial College London, London, UK Japan Science and Technology Agency, ERATO Human Receptor Crystallography Project, Kyoto, Japan Search for more papers by this author Simon Newstead Corresponding Author Simon Newstead Department of Biochemistry, University of Oxford, Oxford, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Nicolae Solcan Nicolae Solcan Department of Biochemistry, University of Oxford, Oxford, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author Jane Kwok Jane Kwok Department of Biochemistry, University of Oxford, Oxford, 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 Alexander D Cameron Alexander D Cameron Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author David Drew David Drew Division of Molecular Biosciences, Imperial College London, London, UK Search for more papers by this author So Iwata So Iwata Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK Division of Molecular Biosciences, Imperial College London, London, UK Japan Science and Technology Agency, ERATO Human Receptor Crystallography Project, Kyoto, Japan Search for more papers by this author Simon Newstead Corresponding Author Simon Newstead Department of Biochemistry, University of Oxford, Oxford, UK Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Author Information Nicolae Solcan1,2, Jane Kwok1, Philip W Fowler1, Alexander D Cameron2,3,4, David Drew4, So Iwata2,3,4,5 and Simon Newstead 1,2,3,6 1Department of Biochemistry, University of Oxford, Oxford, UK 2Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK 3Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK 4Division of Molecular Biosciences, Imperial College London, London, UK 5Japan Science and Technology Agency, ERATO Human Receptor Crystallography Project, Kyoto, Japan 6Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan *Corresponding author. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Tel.: +44 1865 613319; Fax: +44 1865 613201; E-mail: [email protected] The EMBO Journal (2012)31:3411-3421https://doi.org/10.1038/emboj.2012.157 There is a Have you seen? (August 2012) associated 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 Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family. Introduction Peptide transport is the main route through which the body absorbs and retains dietary protein and as such plays an important role in human physiology (Steinhardt and Adibi, 1986; Matthews, 1991). Ingested protein is absorbed across the intestinal brush border membrane in the form of di- and tri-peptides through the action of the integral membrane peptide transporter, PepT1 (Fei et al, 1994; Liang et al, 1995; Leibach and Ganapathy, 1996). A similar process occurs at the renal epithelium, where PepT2 re-absorbs small peptides from the glomerular filtrate (Daniel and Rubio-Aliaga, 2003; Biegel et al, 2006). Both PepT1 and PepT2 recognize a diverse library of peptide substrates, which include most proteinogenic di- and tri-peptides (Matthews, 1975), in keeping with their function as major nutrient transporters. They also recognize and play an active role in the oral absorption and renal retention of many drug compounds with a steric resemblance to peptides, which include the commonly prescribed β-lactam antibiotics (Luckner and Brandsch, 2005; Anderson and Thwaites, 2010). A detailed understanding of the structure, transport mechanism and conformational changes of PepT1 and PepT2 would therefore substantially improve efforts to utilize these transporters for improved drug delivery, distribution and retention (Brandsch, 2009). PepT1 and PepT2 belong to the solute carrier (SLC) 15 gene family and phylogenetically form part of the much larger proton-dependent oligopeptide transporter, or POT family (TC 2.A.17) that is widely distributed within prokaryotes and eukaryotes (Daniel et al, 2006). They are all proton (H+)-driven symporters, using the inwardly direct proton electrochemical gradient (ΔμH+) to drive the uptake of peptides across cell membranes (Daniel and Kottra, 2004). The high degree of sequence conservation between prokaryotic and eukaryotic members (Supplementary Figure S1) indicates that they operate through a conserved mechanism (Daniel et al, 2006). This conclusion is supported by several biochemical studies on prokaryotic PepT1 and PepT2 homologues (Fang et al, 2000; Weitz et al, 2007; Harder et al, 2008; Ernst et al, 2009; Jensen et al, 2011; Malle et al, 2011). Structurally, the POT family sits within the larger and functionally diverse major facilitator superfamily (MFS) of secondary active transporters (Pao et al, 1998) that typically contain 12, but sometimes 14, trans-membrane (TM) helices. Crystal structures from several MFS transporters reveal a common fold consisting of two 6-TM bundles that assemble together in the membrane to form a ‘V’-shaped transporter with a central substrate-binding site formed between the two bundles (Hirai et al, 2002; Abramson et al, 2003; Huang et al, 2003; Yin et al, 2006; Dang et al, 2010). Based on these structures and numerous biophysical studies on LacY, the lactose permease from Escherichia coli (Smirnova et al, 2011), a general model of transport within the MFS has been proposed. This broadly describes transport occurring through the movement of each bundle around a central binding site and is commonly referred to as the rocking bundle mechanism (Law et al, 2008). Recently, the first crystal structure of a POT transporter, PepTSo, was determined (Newstead et al, 2011), revealing a novel asymmetrical occluded conformation, previously unobserved for this class of transporter. An intracellular gate that appears to be conserved among many POT family members was observed restricting the exit of a bound ligand from the intracellular side of the transporter. The structure and mechanism of these gates within the general rocking bundle model of transport is currently not well defined for the MFS, largely due to the absence of crystal structures representing different stages of the transport cycle from the same family. Here, we report the crystal structure of a second POT family transporter from the bacterium Streptococcus thermophilus, PepTSt refined at 3.3 Å resolution. In contrast to the occluded structure of PepTSo, the structure of PepTSt reveals an inward facing conformation that provides significant new insight into the release mechanism of the intracellular gate and the role of conserved salt bridge interactions in orchestrating structural changes during transport. We further identify key residues involved in proton binding and regulating peptide affinity within the binding site, which combined with the structural data provide a more detailed structural model for peptide transport within the POT family. Results Structure, kinetics and substrate specificity of PepTSt The structure of PepTSt was solved using multiple isomorphous replacement with anomalous scattering (MIRAS) using mercury derivatized crystals and seleno-L-methionine incorporated protein (for further details, see Supplementary data). The structure was refined at 3.3 Å resolution to a final Rfactor of 27.3% and Rfree of 28.9% (Table I). PepTSt adopts the canonical MFS fold with helices H1–H6 forming the N-terminal bundle and helices H7–H12 the C-terminal bundle and represents an inward facing conformation for the POT family (Figure 1A). The two bundles adopt similar structures and superimpose with a root mean square deviation (r.m.s.d.) of 2.7 Å over 153 Cα atoms. The peptide-binding site is located at the apex of an elongated hydrophilic cavity that opens outwards from the interior towards the intracellular side of the membrane (Figure 1B). The cavity has overall dimensions of ∼14 Å × 13 Å × 20 Å. The extracellular side of the binding site by comparison is tightly sealed, through the close packing of helices H1 and H2 against H7 and H8 forming an occlusion that hereafter we refer to as the extracellular gate. In addition to the two 6-TM bundles, PepTSt contains two other helices, HA and HB, which are inserted within the intracellular loop connecting helices H6 and H7 and pack to one side of the transporter (Supplementary Figure S3). Similar helices were observed in the related POT family member, PepTSo (Newstead et al, 2011). The absence of these helices in the fungal, plant and metazoan protein sequences suggests that they do not form part of a conserved transport mechanism and short 100 ns atomistic simulations indicate that these helices move semi-independently from the ‘core’ 12 TM MFS fold (Supplementary Figure S4). Figure 1.PepTSt structure reveals an inward open conformation. (A) Overall structure of PepTSt viewed from the extracellular side of the molecule. The 12-TM MFS fold is coloured blue to red with helices HA and HB coloured grey. The helices are labelled. The right-hand image shows a view in the plane of membrane with approximate dimensions of the molecule. (B) Slab through the surface electrostatic potential of PepTSt viewed in the plane of membrane to highlight the extracellular gate, central peptide-binding site and intracellular gate. (C) Peptide transport by PepTSt is driven by an inwardly directed proton gradient and selective for L di- and tri-peptides. ‘No competitor’ refers to the condition where only 3H di-alanine peptide was present in the external buffer, without additional cold peptides. ‘No protein’ refers to empty liposomes. ‘CCCP’ refers to addition of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine to the external buffer. Error bars indicate the standard deviations from triplicate experiments. (D) PepTSt displays higher affinity for hydrophobic di-peptides and can discriminate based on peptide size and charge. Download figure Download PowerPoint Table 1. Data collection, phasing and refinement statistics for PepTSt Native (high) Native (low) MMC-1a MMC-2 Se Data collection Space group P212121 P212121 P212121 P212121 P212121 Cell dimensions a, b, c (Å) 89.6, 113.1, 215.3 90.4, 113.6, 223.4 90.7, 113.8, 222.5 90.7, 113.7, 224.9 90.6, 113.6, 222.61 α, β, γ (deg) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) 107–3.3 (3.4–3.3) 74–4.0 (4.1–4.0) 70–4.5 (4.6–4.5) 70–5.9 (6.0–5.9) 90–4.5 (4.6–4.5) Rmergeb 10.1 (111.2) 8.1 (85.0) 9.4 (82.2) 6.4 (60.3) 6.6 (47.9) I/σI 5.1 (1.1) 9.6 (1.9) 10.9 (2.7) 16.8 (2.0) 9.0 (2.7) Completeness (%) 99.3 (99.9) 94.0 (97.4) 97.2 (96.2) 98.5 (93.6) 96.8 (90.9) Redundancy 3.5 (3.5) 3.0 (3.0) 6.7 (6.5) 6.8 (6.9) 3.5 (3.5) Rcullis (%)Isomorphous/anomalous 90.3/95.3 109.2/93.5 94.6/90.8 Phasing powercIsomorphous/anomalous 0.583/0.459 0.345/0.709 0.658/0.646 Refinement Resolution (Å) 29–3.3 No. of reflections 27 943 Rwork/Rfree 27.3/28.9 No. of atoms 13 898 Protein r.m.s.d. Bond lengths (Å) 0.01 Bond angles (deg) 0.98 a For details on derivatization, see Supplementary data. b The last shell Rmerge is high for some of the derivative data due to severe anisotropy in the diffraction images. c Phasing power=rms (∣FH∣/((FH+FP)−(FPH))). To establish PepTSt as a model for understanding transport within the wider POT family, purified protein was reconstituted into a proton-driven proteoliposome system (for further details, see Supplementary data). Maximal transport of the peptide analogue glycylsarcosine was observed at pH 6.5 with an apparent affinity constant, KMapp, of 9.3 mM and a maximal rate of uptake, Vmax, of 631 nmol min−1 mg−1 PepTSt (Supplementary Figure S5), consistent with the values obtained for DtpT from Lactococcus lactis using a similar assay technique (Fang et al, 2000). The size and charge preference for peptides was also investigated (Figure 1C). As observed for the mammalian PepT1 protein (Fei et al, 1994), only di- and tri-peptides competed effectively for uptake of the 3H di-alanine reporter peptide, with an approximate 10-fold increase in affinity for di-alanine (IC50 0.03±0.01 mM) over that for tri-alanine (0.4±0.05 mM; Figure 1D). The affinity for hydrophobic di-peptides is higher than for charged di-peptides, ranging from 0.05±0.01 mM for di-phenylalanine to 2±0.2 mM for di-lysine. Di-glutamate competes as well for uptake as tri-alanine, suggesting that the peptide-binding site effectively discriminates between positive and negatively charged side chains, which is also the case for the mammalian homologues (Eddy et al, 1995). Also consistent with mammalian peptide transporters, transport by PepTSt appears to be stereospecific, with no discernable inhibition by D-Ala-D-Ala. The extracellular gate is stabilized by conserved salt bridge interactions Alternating access within many secondary active transporters occurs through conformational changes that result in the ligand binding sites being alternatively exposed to either side of the membrane. These changes are often described in terms of the opening and closing of gates, which are often local areas of the structure that move in response to ligand or ion binding (reviewed in Forrest et al, 2011). Access to the peptide-binding site from the extracellular side of the membrane in PepTSt will require a substantial conformational change within the extracellular gate. Two salt bridge interactions can be identified that facilitate the close packing of the gate helices in the structure (Figure 2A), namely Arg53 (H1) with Glu312 (H7) and Arg33 (H1) with Glu300 (H7). The Arg53–Glu312 salt bridge is distal from the peptide-binding site, being located at the extracellular side of the transporter. Mutation of Arg53 to alanine reduced the proton-driven uptake of di-alanine by 50% compared with the WT protein, whereas a similar mutation of Glu312 reduced uptake by 70%, similar to a double alanine mutant (Figure 2B); however, a charge swapped mutant only showed a 20% reduction in transport. Taken together, these results suggest the Arg53–Glu312 salt bridge plays a supportive rather than essential role during transport. In contrast, alanine mutations at the second salt bridge, between Arg33 and Glu300, that is positioned next to the peptide-binding site had a substantial effect on transport. The mutation of Glu300 to alanine abolished uptake in both the proton-driven and peptide-driven counterflow assay, whereas transport in the Arg33 mutant was only abolished in the proton-driven assay (Figure 2C). This is an intriguing result, indicating that Arg33 is important for coupling peptide transport to the inwardly directed proton gradient whereas Glu300 appears to affect peptide recognition and/or structural movements during transport; this role is consistent with the prominent position of Glu300 within the extracellular gate. However, an additional role for Glu300 in proton coupling should not be disregarded, as this may be required for subsequent peptide binding, a situation that would give similar results to those observed. Nevertheless, as discussed below, we propose these interactions are important in orchestrating alternating access within the POT family. Figure 2.Salt bridges facilitate closure of the extracellular gate. (A) Two prominent salt bridge interactions (Arg53–Glu312: distance ∼2.9 Å and Arg33–Glu300: distance ∼3.8 Å) are observed facilitating the close packing between helix hairpins H1–H2 and H7–H8 in the extracellular gate. Residues forming the central peptide-binding site are shown in yellow and the salt bride interactions in red. Helices and residues are labelled. (B) Effect of mutations in the Arg53–Glu312 salt bridge on proton-driven 3H di-alanine uptake. (C) Effect of mutations in the Arg33 and Glu300 salt bridge on proton driven (left-black bars) and peptide-driven counterflow transport (right-blue bars). Details of the counterflow experiments can be found in Supplementary data. Error bars indicate the standard deviations from triplicate experiments. Download figure Download PowerPoint Residues involved in proton binding and the role of the ExxERFxYY motif Contributing to the central cavity is a conserved sequence motif (ExxERFxYY) on helix H1 that is found in all POT family members studied to date (Supplementary Figure S1; Daniel et al, 2006). The functional significance of this motif however remains ambiguous. The arginine and tyrosine residues from this motif are positioned close to the ligand seen in the binding pocket of the occluded structure of PepTSo (Newstead et al, 2011). To address the functional role of this motif in PepTSt, Glu22, Glu25, Arg26, Tyr29 and Ty30 (Figure 3A) were mutated sequentially to alanine (Supplementary Table 1); however, as the Arg26Ala mutant did not express this was further substituted for lysine. We interpret our data to indicate the ExxERFxYY motif is important for proton binding during transport, as the alanine mutants showed measurable uptake only under counterflow conditions (Figure 3B and C). The exception was Tyr29, which retained 75% of WT levels and as shown below is likely to function in determining peptide specificity. Figure 3.Proton binding and peptide specificity reside predominantly within the N-terminal domain of PepTSt. (A) Peptide-binding site as viewed from the plane of the membrane showing the ExxERFxYY motif on helix H1. Residues are coloured according to their predicted role, with proton binding (green), peptide specificity (pink) and transport (yellow). (B) Effect of substitutions within the peptide-binding site on proton-driven uptake. (C) Effect of equivalent substitutions on peptide driven counterflow uptake. (D) Effect of phenylalanine and alanine substitutions at Tyr29 and Tyr68 on the ability of different L-isomer peptides to compete for uptake of di-alanine in proton-driven uptake. (E) Change in IC50 values upon substitution of Tyr29 and Tyr68 to phenylalanine. The IC50 values for the different peptides were calculated as described in Supplementary data. Error bars indicate the standard deviations from triplicate experiments. Download figure Download PowerPoint In the structure, Glu25 and Arg26 interact through a salt bridge at a distance of 2.5–2.8 Å, which is positioned close ∼3.2 Å to Glu22 on helix H1 (Figure 3A; Supplementary Figure S2A). To test the functional significance of this salt bridge, we substituted Glu22 and Glu25 to glutamine. As with the alanine substitutions described above, both Glu22Gln and Glu25Gln variants were inactive under proton-driven uptake (Figure 3B) but retained activity under counterflow (Figure 3C), providing further support for a role in the proton coupling mechanism and suggesting that the chemistry of the salt bridge between Glu25 and Arg26 is an important component of the ExxERFxYY motif in PepTSt. Positioned close ∼3.8 Å to Arg26 in the peptide-binding site is a conserved lysine on helix H4, Lys126 (Figure 3A). Lys126 is absolutely conserved throughout the POT family (Supplementary Figure S1) and holds a prominent position within the binding sites of both PepTSt and PepTSo (Newstead et al, 2011). Mutation of Lys126 to alanine only abolished proton-driven uptake (Figure 3B and C), providing strong evidence for proton binding during transport. Interestingly, the primary amine group is absolutely required as substitution for arginine, histidine or glutamine all abolished proton-driven uptake, but left counterflow unaffected (Supplementary Figure S6). The close positioning of Lys126 to the ExxERFxYY motif in the structure suggests that a functional interaction during transport is possible, potentially acting to regulate the proton coupling we observe. Another striking feature of the central cavity is the cluster of three glutamates on the C-terminal half of the molecule (Figure 3A). Glutamates are likely candidates for proton binding and suggested to play such a role in the fucose and lactose permeases (Dang et al, 2010; Kaback et al, 2011). Two of the glutamates in PepTSt, Glu299 and Glu300 are found on H7, with Glu300 forming part of the extracellular gate, as discussed above. Substitution of Glu299 to alanine, glutamine or the conservative aspartic acid resulted in a protein too unstable to be produced (Supplementary Table 1). Although an acidic residue is highly conserved at the equivalent position to Glu300 on H7, the preceding residue is often either phenylalanine or tyrosine (Supplementary Figure S1), suggesting that Glu299 is playing a structural or stabilizing role unique to PepTSt. The third glutamate, Glu400, is situated on H11 and close ∼3.2 Å to a conserved asparagine on H8, Asn328. Of these glutamates, only Glu400 is strictly conserved within the POT family. The equivalent glutamate to Glu400 in human PepT1 (Glu595) is attributed to binding the N-terminus of peptide substrates (Meredith et al, 2000), a role supported in biochemical studies on the E. coli peptide transporter, YjdL (Jensen et al, 2011). In PepTSt, the Glu400Ala mutation failed to express; similar mutations in human PepT1 and YjdL express but were not functional (Bolger et al, 1998; Jensen et al, 2011). Surprisingly, the conservative Glu400Asp mutation also abolished proton-driven and counterflow uptake (Figure 3C), indicating a requirement for the increased length of the glutamate side chain in PepTSt. Role of conserved tyrosine's in determining peptide binding affinities The peptide-binding site contains three prominent tyrosine side chains, Tyr29 and Tyr30, contributed from helix H1, and Tyr68 from helix H2 (Figure 3A). A similar cluster of tyrosine side chains was observed in the binding site of PepTSo (Newstead et al, 2011) and suggested to play a role in mediating peptide specificity, as shown for rabbit PepT1 (Pieri et al, 2009). The hydroxyl groups of both Tyr29 and Tyr68 do not contribute to proton binding, as their mutation to phenylalanine had little impact on proton-driven uptake compared with WT (Figure 3B). The Tyr29Ala mutant did show reduced uptake in counterflow, however, demonstrating that the ExxERFxYY motif has additional roles to proton binding during transport. To investigate the function of Tyr29 and Tyr68 further we carried out a series of competition experiments with a library of di- and tri-peptides (Figure 3D). The phenyalanine mutants showed distinct changes in their affinity for di-Glu and tri-Ala peptides compared with the WT protein, with the alanine mutants losing their ability to transport these peptides altogether while still retaining affinity for di-Phe and di-Ala peptides (Figure 3D). This difference in the observed affinity between peptides, in particular for the alanine mutations, suggests that Tyr29 and Tyr68 are important in determining peptide affinity. To quantify the contribution made by Tyr29 and Tyr68 to peptide affinity, IC50 values for these peptides were calculated for each of the phenylalanine mutants (Figure 3E). The Tyr29Phe mutant had a decreased affinity for tri-alanine, IC50 of 1.4 mM compared with 0.4 mM for the WT, while still retaining WT affinity levels for di-Glu. The Tyr68Phe mutant displays a decreased affinity for di-Glu, IC50 values of 1.63 mM compared with 0.56 mM for the WT protein while retaining the same affinity for tri-alanine. Taken together, these results identify helices H1 and H2 within the PepTSt as critical sites of interaction with the peptide during transport. Comparison between inward open and occluded POT family transporters reveals a structural movement that opens the intracellular gate Comparing PepTSt and PepTSo, helices H1–H6 superimpose with an r.m.s.d. of 1.6 Å for 167 Cα atoms, adopting a similar arrangement between the two structures (Supplementary Figures S1 and S7). The main difference occurs within the C-terminal bundle, where helices H7–H12 superimpose with an r.m.s.d. of 2.3 Å for 127 Cα atoms. In the occluded structure of PepTSo, an intracellular gate constructed from conserved side chain interactions between helices H4–H5 and H10–H11 prevents the bound ligand from exiting the binding site (Newstead et al, 2011). Comparison with the inward open structure of PepTSt shows the cytoplasmic halves of H7, H10 and H11 swing away from helices H4–H5 resulting in the release of this gate, opening the peptide-binding site to the intracellular side of the membrane (Figure 4A). Unexpectedly, opening of this gate does not require movement of the entire H10–H11 helix hairpin, as previously conjectured (Newstead et al, 2011), but appears to be localized towards the cytoplasmic halves of these helices and consist of a hinge like movement at the apex of the H10–H11 hairpin (Figure 4A). Specifically, the gate opens due to bending at Gly407 and Trp427 on helices H10 and H11, respectively. This results in the intracellular ends of these helices moving ∼13 Å away from their respective positions in the occluded conformation. This movement appears coordinated with respect to helix H7, which straightens in PepTSt providing space for H10 and H11 to move. Gly407 and Trp427 are located at the same point within the H10–H11 helix hairpin, effectively forming a hinge or pivot point, which would control whether the intracellular gate is open or closed between the occluded and inward facing conformation. Indeed, substitution of either Gly407 or Trp427 to alanine abolished both proton-driven and counterflow uptake (Figure 4B). A Trp427Phe substitution however did not adversely affect transport in either assay, implying a bulky hydrophobic residue at this position is sufficient for normal functioning of the gate. Of note is that glycine residues are found at equivalent positions to Gly407 in GlpT (Lemieux et al, 2004), FucP (Dang et al, 2010), EmrD (Yin et al, 2006) and LacY (Abramson et al, 2003), implying a wider significance for fl
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