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• W4232085364 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues. https://doi.org/10.7554/eLife.02283.001 eLife digest Molecules of glutamate can carry messages between cells in the brain, and these signals are essential for thought and memory. Glutamate molecules can also act as signals to build new connections between brain cells and to prune away unnecessary ones. However, too much glutamate outside of the cells kills the brain tissue and can lead to devastating brain diseases. In a healthy brain, special pumps called glutamate transporters move these molecules back into the brain cells, where they can be stored safely. However, when brain cells are damaged—by, for example, a stroke or an injury,—the glutamate stored inside spills out, killing the surrounding cells. This leads to a cascade of dying cells and leaking glutamate, which causes even more damage and slows the recovery. Glutamate transporters ensure that there are more glutamate molecules inside cells than outside. However, it requires energy to maintain this gradient in the concentration of glutamate molecules. The transporters get this energy by moving three sodium ions into the cell with each glutamate molecule, and moving one potassium ion out of the cell. However, it is not clear how these transporters ensure that they move the glutamate molecules and the sodium ions at the same time. Now, Verdon, Oh et al. have uncovered the 3D structure of a glutamate transporter homologue at each step of the transport process. These structures reveal that, on the outside of the cell membrane, sodium ions attach to the so-called ‘transporter domain’ and make it better able to bind glutamate. The transporter domain then carries the sodium ions and glutamate through the cell membrane and releases them into the cell. Verdon, Oh et al. suggest that a potassium ion then binds to the empty transport domain, stabilizing it into a more compact shape that easily makes the return trip to the outside of the cell. Most experiments on glutamate transporters, including the work of Verdon, Oh et al., are carried out on model proteins taken from bacteria. An important challenge for the future will be to obtain structural information on human glutamate transporters, as these could be therapeutic targets for the treatment of various neurological conditions. https://doi.org/10.7554/eLife.02283.002 Introduction Glutamate transporters, or excitatory amino acid transporters (EAATs), reside in the plasma membranes of glial cells and neurons, where they catalyze the re-uptake of the neurotransmitters glutamate and aspartate (L-asp) (Danbolt, 2001). EAATs terminate neurotransmission events supporting memory formation and cognition, and also prevent excitotoxicity caused by overstimulation of glutamate receptors. Dysfunction of EAATs is linked to neurological disorders, poor recovery from stroke and traumatic brain injuries (Yi and Hazell, 2006; Sheldon and Robinson, 2007; Kim et al., 2013). To maintain steep trans-membrane glutamate gradients, EAATs transport one substrate molecule together with three sodium ions (Na+) and one proton. After their release into the cytoplasm, counter-transport of one potassium ion (K+) resets the transporter for the next cycle (Zerangue and Kavanaugh, 1996; Levy et al., 1998; Owe et al., 2006). Key mechanistic and structural insights into this family of transporters come from studies on an archaeal homologue from Pyrococcus horikoshii, GltPh (Figure 1—figure supplement 1), which symports L-asp together with three Na+ ions (Groeneveld and Slotboom, 2010); however, it shows no dependence on counter-transport of K+ under the conditions tested (Ryan et al., 2009). GltPh, like EAATs, is a homo-trimer (Gendreau et al., 2004; Yernool et al., 2004). Each protomer consists of a central scaffolding trimerization domain and a peripheral transport domain containing the substrate and ion binding sites (Boudker et al., 2007; Reyes et al., 2009). When bound to Na+ and L-asp (‘fully bound’ from here on), each transport domain moves by ∼15 Å across the membrane from an outward- to an inward-facing position, in which the substrate binding site is near the extracellular solution and the cytoplasm, respectively (Reyes et al., 2009). Structurally symmetric helical hairpins, HP1 and HP2, occlude the bound substrate from the solvent and are thought to serve as gates (Boudker et al., 2007; Huang and Tajkhorshid, 2008; Shrivastava et al., 2008; Reyes et al., 2009; DeChancie et al., 2010; Focke et al., 2011; Zomot and Bahar, 2013). Two Na+-binding sites (Na1 and Na2), neither of which directly coordinates the substrate, were identified crystallographically using thallium (Tl+) (Boudker et al., 2007). The location of the third Na+-binding site is being debated (Holley and Kavanaugh, 2009; Huang and Tajkhorshid, 2010; Larsson et al., 2010; Tao et al., 2010; Bastug et al., 2012; Teichman et al., 2012). A highly conserved non-helical Asn310-Met311-Asp312 (NMD) motif interrupts trans-membrane segment (TM) 7 (see below). It lines the back of the substrate- and ion-binding sites and is involved in binding of the ligands (Rosental et al., 2006; Tao et al., 2006; Rosental and Kanner, 2010). The main chain carbonyl oxygen of Asn310 contributes to Na1 site, while the side chain of Met311 protrudes between the substrate, Na1 and Na2 binding sites (Boudker et al., 2007). Symport requires that neither the substrate nor the ions alone are efficiently transported (Crane, 1977). Therefore to traverse the membrane, the transport domains of GltPh and EAATs must be loaded with both Na+ ions and substrate. To complete the transport cycle, the transport domain of GltPh must also translocate readily when it is free of both solutes (apo), while in EAATs it requires binding of a K+ ion. To establish the structural underpinnings of these processes, we determined crystal structures of the outward- and inward-facing states of GltPh in apo and ions-only bound forms (Tables 1, 2 and 3). We find that the apo transport domain shows identical structures when facing outward or inward. While ligand-binding sites are distorted, the domain remains compact, suggesting that it relocates across the membrane as a rigid body, similarly to when it is fully bound (Reyes et al., 2009). Ion binding to Na1 site, located deep in the core of the transport domain, triggers structural changes that are propagated to the extracellular gate HP2, at least in part, by the side chain of Met311 in the NMD motif. Consequently HP2, which in the apo form is collapsed into the substrate binding and Na2 sites, frees the sites, assuming conformations more similar to the conformation observed in the fully bound transporter. We suggest that these Na+-dependent structural changes underlie the high cooperativity of Na+ and substrate binding, which is thought to be one of the key coupling mechanisms (Reyes et al., 2013). Furthermore, in the structure of Na+-bound outward-facing GltPh we observe opening of HP2 tip, which may facilitate L-asp access to its binding site and prevent the inward movement of the Na+-only bound transport domain, as previously suggested (Focke et al., 2011). Remarkably, soaks of apo GltPh crystals in Tl+ reveal new cation-binding sites within the apo-like protein architecture. One such site overlaps with the substrate-binding site. Because binding of a cation to this site would compete with binding of Na+ and the transported substrate, it is well suited to serve as a binding site for a counter-transported ion. We propose that the closed translocation-competent conformation of the transport domain free of Na+ and substrate is intrinsically stable in GltPh but not in EAATs, in which K+ binding at the newly identified site is required, coupling transport cycle completion to K+ counter-transport. Table 1 X-ray crystallographic data and refinement statistics for GltPh-R397A and GltPh-K55C-A364CHg (GltPhin) structures deposited at the PDB https://doi.org/10.7554/eLife.02283.005 GltPhinapoTl+-bound (apo conf.)alkali-freeTl+-bound (bound conf.)Data collection Space groupC2221C2221C2221C2221 Cell dimensions a, b, c (Å)109.93, 201.81, 207.14106.98, 196.56, 206.50106.95, 196.84, 207.48110.83, 200.43, 206.40 α, β, γ (°)90.00, 90.00, 90.0090.00, 90.00, 90.0090.00, 90.00, 90.090.00, 90.00, 90.00 Resolution (Å)100.0–3.25 (3.31–3.25)100.0–3.75 (3.81–3.75)100.0–3.50 (3.56–3.50)100.0–4.0 (4.14–4.0) Rsym or Rmerge10.9 (88.6)14.0 (94.4)8.0 (88.1)16.3 (75.2) I/σI12.3 (1.2)8.95 (1.1)13.5 (1.2)7.9 (1.3) Completeness (%)98.7 (88.1)99.7 (99.8)94.4 (92.7)65.2 (6.5) Redundancy5.6 (2.8)3.8 (3.7)3.3 (3.2)3.4 (3.5)Refinement Resolution (Å)15.0–3.2515.0–3.7515.0–3.515.0–4.0 No. reflections34534215652544611105 Rwork/Rfree22.2/25.823.0/25.726.3/27.825.8/29.6 No. atoms Protein9121911490888985 Ligand/ion3939 B-factors Protein108.5141.8144.2137.2 Ligand/ion135.3170.8214.1102.3 R.m.s. deviations Bond lengths (Å)0.0100.0130.0050.012 Bond angles (°)1.6801.8611.1161.407PDB code4P194P1A4P3J4P6H GltPh-R397AApoNa+-boundNa+/aspartate-boundData collection Space groupP21P31P31 Cell dimensions a, b, c (Å)112.37, 424.42, 113.99110.58, 110.58, 306.92116.96, 116.96, 313.52 α, β, γ (°)90.00, 119.40, 90.0090.00, 90.00, 120.0090.00, 90.00, 120.00 Resolution (Å)100.0–4.00 (4.14–4.00)50.0–3.39 (3.51–3.39)100.0–3.50 (3.63–3.50) Rsym or Rmerge7.8 (62.2)14.0 (>100)8.4 (>100) I/σI9.3 (1.3)13.8 (1.4)10.6 (0.4) Completeness (%)67.9 (13.0)87.3 (12.0)98.1 (96.6) Redundancy1.8 (2.0)11.8 (8.6)4.5 (4.2)Refinement Resolution (Å)20.0–4.012.0–3.4115.0–3.50 No. reflections520684836655613 Rwork/Rfree24.9/26.628.4/29.324.3/26.8 No. atoms Protein352771758018192 Ligand/ionN/A654/12 WaterN/A66 B-factors Protein139.5152.097.1 Ligand/ionN/A145.184.7/86.9 WaterN/A102.6144.6 R.m.s. deviations Bond lengths (Å)0.0100.0100.015 Bond angles (°)1.3931.4681.735PDB code4OYE4OYF4OYG Table 2 Completeness of datasets corrected for anisotropy https://doi.org/10.7554/eLife.02283.006 Tl+-bound GltPhin (bound conformation)Na+-bound GltPh-R397AResolution range (Å)Completeness (%)Resolution range (Å)Completeness (%)100.0–8.6299.350.00–7.3099.68.62–6.8499.97.30–5.79100.06.84–5.97100.05.79–5.06100.05.97–5.4399.95.06–4.60100.05.43–5.0499.94.60–4.27100.05.04–4.7469.64.27–4.02100.04.74–4.5039.24.02–3.82100.04.50–4.3123.63.82–3.6598.64.31–4.1414.43.65–3.5163.04.14–4.006.53.51–3.3912.0 Apo GltPh-R397AResolution range (Å)Completeness (%)100.0–8.6285.08.62–6.8475.66.84–5.9775.55.97–5.4375.35.43–5.0475.25.04–4.7475.84.74–4.5075.34.50–4.3175.44.31–4.1451.74.14–4.0013.0 Table 3 X-ray crystallographic data and refinement statistics for GltPh-R397A and GltPh-K55C-A364CHg structures not deposited at the PDB https://doi.org/10.7554/eLife.02283.007 GltPh-R397AGltPhinTl+-bound (apo conf.)Tl+/Na+ (apo conf.)Tl+/k+ (apo conf.)Data collection Space groupP21C2221C2221 Cell dimensions a, b, c (Å)115.18, 428.53, 116.61108.11, 198.86, 206.34106.59, 198.48, 205.82 α, β, γ (°)90.00, 119.49, 90.0090.00, 90.00, 90.0090.00, 90.00, 90.00 Resolution (Å)30.0–5.0 (5.18–5.00)100.0–4.0 (4.07–4.00)100.0–4.15 (4.22–4.15) Rsym or Rmerge10.9 (>100)15.0 (92.2)13.9 (94.1) I/σI13.8 (1.9)8.9 (1.5)9.2 (1.5) Completeness (%)86.4 (75.1)99.9 (100)94.5 (90.2) Redundancy5.5 (5.8)3.9 (3.9)4.0 (3.9)Refinement Resolution (Å)20.0–5.015.0–4.015.0–4.15 No. reflections347471818415419 Rwork/Rfree22.0/26.528.2/31.728.3/31.2 No. atoms Protein3510791359135 Ligand/ionN/AN/AN/A WaterN/AN/AN/A B-factors Protein223.00183.6194.4 Ligand/ionN/AN/AN/A WaterN/AN/AN/A R.m.s. deviations Bond lengths (Å)0.0080.0060.008 Bond angles (°)1.1861.2661.440 Results Remodeling of the apo transport domain To determine the structure of apo GltPh, we used R397A mutant that shows a drastically decreased affinity for substrate (Figure 1A). When fully bound, GltPh-R397A crystallizes in the outward-facing state, like wild type GltPh, except that L-asp coordination is slightly altered because the mutant is missing the key coordinating side chain of Arg397 (Figure 1B, Figure 1—figure supplement 2; Bendahan et al., 2000; Boudker et al., 2007). These results suggest that R397A is suitable to capture the apo and ions-only bound outward-facing states for their structural characterization. However, removal of Arg397 may affect local electrostatics, potentially altering ion binding; thus these studies should be interpreted with caution. Apo GltPh-R397A also crystallized in an outward-facing conformation that is similar to the structure reported for a close GltPh homologue (Jensen et al., 2013). To obtain an apo inward-facing state, we used GltPh-K55C-A364C mutant trapped in the inward-facing state upon cross-linking with mercury (Reyes et al., 2009) (GltPhin, Figure 1—figure supplement 1). The positions and orientations of the transport domains relative to the trimerization domains remain essentially unchanged in the apo and fully bound forms of GltPh-R397A and GltPhin (Figure 2). In contrast, the conformations of the transport domains themselves differ significantly. Most remarkably, the apo conformations of the transport domain are nearly identical in the outward- and inward-facing states (Figure 3A, Figure 3—figure supplement 1, Figure 3—figure supplement 2A) and are therefore independent of the transport domain orientations and crystal packing environments. Figure 1 with 2 supplements see all Download asset Open asset Substrate binding to GltPh-R397A. (A) Raw binding heat rates measured by isothermal titration calorimetry (top) and binding isotherms (bottom) obtained for GltPh-R397A (left) and wild type GltPh (right) at 25°C in the presence of 100 mM NaCl. The solid lines through the data are fits to the independent binding sites model with the following parameters for GltPh-R397A and wild type GltPh, respectively: enthalpy change (ΔH) of −3.2 and −14.3 kcal/mol; the apparent number of binding sites (n) of 0.8 and 0.7 per monomer; dissociation constant (Kd) of 6.6 µM and 27 nM. Note that L-asp binding to the wild type transporter is too tight at 100 mM NaCl to be accurately measured in this experiment. The binding Kd has been estimated to be ∼1 nM (Boudker et al., 2007). (B) L-asp binding site in GltPh-R397A (left) and wild type GltPh (right). L-asp and residues coordinating the side chain carboxylate are shown as sticks with carbon atoms colored light brown and blue, respectively. Potential hydrogen bonds (distances less than 3.5 Å) between the L-asp side chain carboxylate and transporter residues are shown as dashed lines. Note that Y317, which forms cation-π interactions with guanidium group of R397 in wild type GltPh, interacts directly with L-asp in GltPh-R397A. https://doi.org/10.7554/eLife.02283.008 Figure 2 Download asset Open asset Apo protomer structures. (A) GltPh protomers in the outward-facing state (left) and a GltPhin protomer (right) viewed from within the plane of the membrane. Shown are superimpositions between apo (colors) and fully bound protomers (grey). https://doi.org/10.7554/eLife.02283.011 Figure 3 with 2 supplements see all Download asset Open asset Structures of the apo transport domain. (A) Superimposition of the nearly identical apo transport domains in the outward- and inward-facing states. HP1, HP2, and NMD motif are colored yellow, red, and green, respectively. The remainder of the domain is blue. (B) Superimposition of the fully bound (light colors, PDB accession number 2NWX) and apo GltPhin (dark colors) transport domains. (C) The NMD motif and adjacent TM3. Met311 is shown as sticks, and the light blue spheres indicate the Cα positions for T92 and S93. (D) The HP2-TM8a structural modules in the fully bound (pink) and apo (red) transport domains superimposed on TM8a and HP2b to emphasize the re-orientation of the HP2a. (E) The Na+ and L-asp binding sites in the fully bound (left) and apo forms (right). https://doi.org/10.7554/eLife.02283.012 The conformational differences between fully bound and apo forms of the transport domain include a concerted movement of HP2 and TM8a, which form the extracellular surface of the domain, and local rearrangements at the ligand binding sites, involving HP2, the NMD motif and TM3 (Figure 3B–E, Figure 3—figure supplement 2B, Figure 4). In HP2, the last helical turn of HP2a unwinds, and HP2a together with the loop region at HP2 tip collapse into the substrate and Na2 binding sites. Within the NMD motif, the side chain of Asn310 rotates away from TM3 and partially fills the empty Na1 site, while the side chain of Met311 undergoes an opposite movement, flipping away from the binding sites (Figure 4). Finally, TM3 bends away from the NMD motif, particularly around Thr92 and Ser93 (Figure 3B,C). Notably, these residues together with the side chain of Asn310 form one of the proposed third Na+-binding sites (Huang and Tajkhorshid, 2010; Bastug et al., 2012). Thus, all known ligand-binding sites are distorted in the apo forms (Figure 4). Figure 4 with 1 supplement see all Download asset Open asset Remodeling of L-asp and Na+ binding sites in the apo conformations. Close-up views of the fully bound (left) and apo (right) transport domains at L-asp binding site (top), Na1 and Na2 sites (middle), and one of the proposed locations for the third Na+ binding site (Huang and Tajkhorshid, 2010; Bastug et al., 2012) (dashed circle). https://doi.org/10.7554/eLife.02283.003 The overall structures of the apo transport domain remain as closed and compacted as in the fully bound forms (Figure 4—figure supplement 1). Therefore, we propose that the unloaded transport domains traverse the membrane as rigid bodies as deduced previously for the fully loaded transport domains (Reyes et al., 2009). Insight into the coupling mechanism In GltPh, cooperative binding of Na+ ions and L-asp is central to tightly coupled transport of the solutes (Reyes et al., 2013). Our structures of the apo and fully bound GltPh suggest that binding of L-asp and Na+ at the Na2 site is coupled because the same structural element, the tip of HP2, contributes to both sites and is restructured upon binding. Thus, structural changes in HP2 upon binding of either L-asp or Na+ ion should greatly favor binding of the other. Met311 in the NMD motif is the only residue that is shared between the Na1 site and the substrate and Na2 sites and also undergoes a conformational change upon ligand binding. To examine whether the structural changes in HP2 upon binding of L-asp and Na+ at the Na2 site could occur independently from those in the NMD motif upon Na+ binding at the Na1 site, we modeled transport domains with HP2 in the bound conformation and the NMD motif in the apo conformation, or vice versa (Figure 5A). In both models, the side chain of Met311 clashes with residues in HP2, suggesting that the conformational changes in HP2 and the NMD motif must be concerted. Figure 5 Download asset Open asset Met311 is key to the allosteric coupling. (A) Structural models combining HP2 bound to L-asp and Na+ at Na2 site with apo conformation of the NMD motif (left), and apo conformation of HP2 with the NMD motif bound to Na+ at Na1 site (right). Met311 and clashing residues in HP2 are shown as sticks and transparent spheres. (B) The dependence of L-asp dissociation constant, Kd, on Na+ activity plotted on a log–log scale for mutants within the context of GltPhin (left) and unconstrained GltPh (right). The data were fitted to straight lines with slopes shown on the graph or to arbitrary lines for clarity. Dashed lines and corresponding slopes correspond to published dependences for GltPhin and GltPh (Reyes et al., 2013). https://doi.org/10.7554/eLife.02283.015 We then mutated bulky Met311 to either another bulky residue, leucine, or to a smaller residue, alanine, which is not expected to experience similar clashes. For these mutants, generated in the context of unconstrained wild type GltPh and inward cross-linked GltPhin, we measured the dependence of L-asp dissociation constant on Na+ concentration (Figure 5B). While this dependence is very steep for the wild type GltPh constructs (Reyes et al., 2013) and nearly as steep for the M311L mutants, it is substantially shallower for the M311A mutants. The most parsimonious interpretation of these results is that M311A mutation reduces binding cooperativity between the substrate and Na+ ions. However, it is also possible, though we think unlikely, that the mutation abrogates ion binding at one or more Na+-binding sites in the tested concentration range (1–100 mM). Mutating the equivalent methionine to smaller residues in EAAT3 also resulted in less steep dependence of the ionic currents on Na+ concentration (Rosental and Kanner, 2010). Based on these results, we hypothesize that Met311 is key to the allosteric coupling between the Na1, L-asp and Na2 sites. Consistently, bulky methionine or leucine residues are found at this position in ∼85% of glutamate transporter homologues. However, it should be noted that methionine is conserved in the Na+-coupled GltPh and EAATs, while a characterized proton-coupled homologue has leucine at this position (Gaillard et al., 1996). Hence, it is possible that the methionine thioether, which is proximal to both Na1 and Na2 sites, plays a direct role in Na+ binding. Our hypothesis further predicts that binding of an ion at Na1 site should prime the transporter to accept its substrate. Therefore, we crystallized GltPh-R397A in the presence of 400 mM Na+, but in the absence of L-asp. We also soaked crystals of apo GltPhin in Tl+, an ion with strong anomalous signal that seems to mimic some aspects of Na+ in GltPh and EAATs (Boudker et al., 2007; Tao et al., 2008). The obtained outward- and inward-facing structures pictured the transport domains in conformations overall similar to those observed in the fully bound transporter: straightened TM3, Met311 pointing toward the binding sites, extended helix in HP2a and HP2 tip raised out of the substrate binding site (Figure 6A–D). Indeed, the structure of Tl+-bound GltPhin is indistinguishable from the fully bound GltPhin and both Na1 and Na2 sites are occupied by Tl+ ions (Figure 6A). The structure of Na+-bound GltPh-R397A differs significantly from the fully bound GltPh-R397A only at the tip of HP2 (Figure 3—figure supplement 2, also see below). The coordinating residues at the Na1 site are correctly positioned and the site is likely occupied by a Na+ ion. The Na2 site still shows a distorted geometry: the last helical turn of HP2a points away from the site due to the altered conformation of the tip of HP2 (Figure 6C). Collectively, our results demonstrate that binding of the coupled ions, notably at the Na1 site, is sufficient to trigger isomerization of the transport domain from the apo conformation to the bound-like conformation. The energetic penalty associated with this isomerization likely explains why Na+ ions alone bind weakly to the transporter (Reyes et al., 2013). This experimental observation contrasts with highly favorable calculated binding energies (approximately −10 kcal/mol for Na1) that were obtained using fully bound protein conformation and where the reference ion-free state is the same as the bound state (Larsson et al., 2010; Bastug et al., 2012; Heinzelmann et al., 2013). Figure 6 with 3 supplements see all Download asset Open asset Structures of ions-only bound transport domain. (A) Superimposition of the fully bound transport domains (grey) and Tl+-bound GltPhin transport domain in the bound-like conformation (colors), with the averaged anomalous difference Fourier map contoured at 8σ (cyan mesh). (B) Superimposition of the fully bound (grey) and Na+-only bound GltPh-R397A (colors) transport domains. (C) Na+ and L-asp binding sites with fully-bound structure shown in white and Na+-bound structure in colors. Hinge glycine residues are shown as spheres. The modeled Na+ ion in Na1 site is pink. (D) Superimposition of the HP2-TM8 in the fully bound transport domain (grey) and in GltPh-R397A bound to Na+ only (colors), showing similar conformations of HP2a. (E) WebLogo representation of the consensus sequence and relative abundance of residues in HP2 tip. (F) Surface representation of the transport domain of GltPh-R397A bound to Na+ only showing access to the substrate-binding site. L-asp was placed into the binding site for reference. https://doi.org/10.7554/eLife.02283.016 Na+-mediated gating in the outward-facing state The structure of the Na+-only bound GltPh-R397A shows HP2 in a conformation overall similar to that observed in the fully bound transporter, but with an opened tip (Figure 6B–D,F, Figure 6—figure supplement 1). This opening is smaller than the opening observed previously in the structure of GltPh in complex with the blocker L-threo-β-benzyloxyaspartate (Figure 6—figure supplement 2; Boudker et al., 2007), and it is hinged at two well-conserved glycine residues at positions 351 and 357 (Figure 6C). Interestingly, among the nine amino acids forming the tip in GltPh (residues 351 to 359), five are glycines in the consensus sequence generated for the glutamate transporter family, although not all are present in each homologue (Figure 6E, Figure 6—figure supplement 3). We suggest that the glycines support the structural flexibility of the HP2 tip in all members of the family, but that the structural specifics of the tip opening may vary among homologues. To test whether the trans-membrane movement of the transport domain is possible when the tip of HP2 is opened, we modeled the open tip conformation in the context of the previously reported early transition intermediate structure (Figure 7; Verdon and Boudker, 2012). In this structure, the transport domain tilts towards the trimerization domain but does not yet undergo a significant translation toward the cytoplasm. We find that such intermediate state with the opened tip of HP2 can be achieved without major steric clashes, while further progression of the transport domain to the inward-facing position could be impeded because the tip is likely to clash with TM5 in the trimerization domain (Figure 7B). Also in the inward-facing state HP2 is packed against the trimerization domain and cannot open in a manner observed in the outward-facing state. Consistently, HP2 is closed in GltPhin bound to Tl+ (Figure 6A). Figure 7 Download asset Open asset Modeled Na+-bound early transition intermediate between the outward- and inward-facing states. (A) Surface representations of the protomer in the fully bound intermediate state (PDB code 3V8G) (left), and the modeled Na+-bound intermediate with an open HP2 tip (right) viewed from the extracellular space (top). The model reveals no clashes, suggesting that the observed opening of HP2 is structurally compatible with the intermediate orientation of the transport domain. The arrows indicate the point of access to the domain interface with potentially increased solvent accessibility. (B) Side views of thin cross-sections of the closed fully bound (left) and open Na+-only bound (right) intermediate state. The protomers are sliced normal to the membrane plane, as indicated by the dashed lines in A. https://doi.org/10.7554/eLife.02283.020 Therefore, opening of the HP2 tip upon Na+ binding in the outward-facing state may serve as a structural mechanism preventing uncoupled uptake of Na+ ions. We suggest that the structural changes in the NMD motif and HP2 that are triggered upon Na+ binding at the Na1 site may lead to the loss of direct interactions between the tip of HP2 and the rest of the transport domain, resulting in tip opening. Subsequent binding of L-asp and Na+ at the Na2 site is then required to provide compensatory interactions, allowing HP2 tip to close. Similar conformational behavior has been observed for transporters with the LeuT fold: when bound to Na+ ions only, substrate binding sites are open to the extracellular solution, and substrate binding is required for occlusion (Weyand et al., 2008" @default.
• W4232085364 created "2022-05-12" @default.
• W4232085364 date "2014-02-19" @default.
• W4232085364 modified "2023-09-26" @default.
• W4232085364 title "Decision letter: Coupled ion binding and structural transitions along the transport cycle of glutamate transporters" @default.
• W4232085364 doi "https://doi.org/10.7554/elife.02283.028" @default.
• W4232085364 hasPublicationYear "2014" @default.
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