Coupled ion binding and structural transitions along the transport cycle of glutamate transporters

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. DOI: http://dx.doi.org/10.7554/eLife.02283.001

Symport requires that neither the substrate nor the ions alone are efficiently transported (Crane, 1977). Therefore to traverse the membrane, the transport domains of Glt Ph and EAATs must be loaded with both Na + ions and substrate. To complete the transport cycle, the transport domain of Glt Ph 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 Glt Ph 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 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. DOI: 10.7554/eLife.02283.002  201.81, 207.14 106.98, 196.56, 206.50 106.95, 196.84, 207.48 110.83, 200.43, 206 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 Glt Ph 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 Glt Ph 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 translocationcompetent conformation of the transport domain free of Na + and substrate is intrinsically stable in Glt Ph but not in EAATs, in which K + binding at the newly identified site is required, coupling transport cycle completion to K + counter-transport.

Remodeling of the apo transport domain
To determine the structure of apo Glt Ph , we used R397A mutant that shows a drastically decreased affinity for substrate ( Figure 1A). When fully bound, Glt Ph -R397A crystallizes in the outward-facing state, like wild type Glt Ph , 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.  and binding isotherms (bottom) obtained for Glt Ph -R397A (left) and wild type Glt Ph (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 Glt Ph -R397A and wild type Glt Ph , 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 (K d ) 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 K d has been estimated to be ∼1 nM (Boudker et al., 2007). (B) L-asp binding site in Glt Ph -R397A (left) and wild type Glt Ph (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 Glt Ph , interacts directly with L-asp in Glt Ph -R397A. DOI: 10.7554/eLife.02283.008 The following figure supplements are available for figure 1: However, removal of Arg397 may affect local electrostatics, potentially altering ion binding; thus these studies should be interpreted with caution. Apo Glt Ph -R397A also crystallized in an outwardfacing conformation that is similar to the structure reported for a close Glt Ph homologue (Jensen et al., 2013). To obtain an apo inward-facing state, we used Glt Ph -K55C-A364C mutant trapped in the inward-facing state upon cross-linking with mercury (Reyes et al., 2009) (Glt Ph in , 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 Glt Ph -R397A and Glt Ph in (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. 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).
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 Glt Ph , 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 Glt Ph 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.
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 Glt Ph and inward cross-linked Glt Ph in , we measured the dependence of L-asp dissociation constant on Na + concentration ( Figure 5B). While this dependence is very steep for the wild type Glt Ph 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 . 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 Glt Ph 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 Glt Ph -R397A in the presence of 400 mM Na + , but in the absence of L-asp. We also soaked crystals of apo Glt Ph in in Tl + , an ion with strong anomalous signal that seems to mimic some aspects of Na + in Glt Ph 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 Glt Ph in is indistinguishable from the fully bound Glt Ph in and both Na1 and Na2 sites are occupied by Tl + ions ( Figure 6A). The structure of Na + -bound Glt Ph -R397A differs significantly from the fully bound Glt Ph -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). The structure of the Na + -only bound Glt Ph -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 Glt Ph 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 Glt Ph (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 Figure 5. 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, K d , on Na + activity plotted on a log-log scale for mutants within the context of Glt Ph in (left) and unconstrained Glt Ph (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 Glt Ph in and Glt Ph (Reyes et al., 2013). DOI: 10.7554/eLife.02283.015 Research article  Figure 6A).
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;Krishnamurthy and Gouaux, 2012).
We do not see a transition into an open conformation in the inward-facing Glt Ph in bound to Tl + ions ( Figure 6A). This may be because Tl + ions do not faithfully mimic Na + ions and fail to induce an open state or it may be because Na + bound inward-facing state is, indeed, closed. This latter possibility does not contradict the requirements of symport because the measured dissociation constant for Na + ions in the inward-facing state (250 mM) (Reyes et al., 2013), is far above Na + concentration in the cytoplasm (10 mM) and therefore, Na + -bound inward-facing state is not expected to be significantly populated.
We and others have proposed that transition intermediates mediate fluxes of polar solutes, including anions, because potentially hydrated cavities form in such intermediates at the interface between the trimerization and transport domains (Stolzenberg et al., 2012;Verdon and Boudker, 2012;Li et al., 2013). Interestingly, because the tip of HP2 forms part of this interface in the fully bound intermediate state of the transporter, opening of the tip in the Na + -only bound form may increase solvent accessibility to the interface ( Figure 7A).

New cation-binding sites
While soaking apo Glt Ph in crystals in Tl + solutions, we observed that only in approximately one third of crystals Tl + ions bound to the Na1 and Na2 sites, inducing transition from apo-to bound-like conformation as described above. In the majority of the crystals, we observed no conformational changes of the transport domain and Tl + ions incorporated at two previously uncharacterized sites (Figure 8), within the small cavities that remain under the collapsed HP2 (Figure 8-figure supplement 1). One site, termed Na2', involves residues of HP2 and TM7a that form the Na2 site, but in a different ion coordinating geometry due to the conformational difference in HP2 ( Figure 8B). The second site, termed Ct, overlaps with the L-asp binding site and is formed by the side chains of highly conserved Asp394 and Thr398 in TM8 and main chain carbonyl oxygen atoms of HP1 and HP2 ( Figure 8B,C). Tl + soaks of the outward-facing apo Glt Ph -R397A also showed no conformational changes of the transport domain, with Tl + binding at the Ct site, but not at the Na2' site ( Figure 8A). The ion selectivity of the Ct site remains ambiguous, because neither 300 mM K + nor 10 mM Na + efficiently inhibited incorporation of Tl + (150 mM) at this site in Glt Ph in (Figure 8figure supplement 2). Crystals deteriorated at higher Na + concentrations. In contrast, the Na2' site seems to show a preference for Na + , which even at low concentration (10 mM) interfered significantly with Tl + binding. The functional relevance of these sites is speculative at present. However, it is remarkable that the Ct site is positioned exactly at the same place as the amino group of the bound L-asp and share several coordinating moieties. Therefore, binding of a cation at the Ct site and binding of the substrate are mutually exclusive. Because the Ct site is observed only in the apo-like conformation, cation binding at this site would also inhibit the transition into the bound-like conformation upon Na + binding at the Na1 site. Finally, the Ct site is observed in both the inward-and outward-facing states, suggesting that the apo-like transport domain could carry the ion across the membrane. These are the exact properties  Figure 8. Continued on next page expected for the K + -binding site in EAATs. Moreover, it has been previously proposed that K + binds to EAATs at a similar position (Holley and Kavanaugh, 2009). Most remarkably, in an insect K +independent dicarboxylate transporter, an asparagine to aspartate mutation at the position equivalent to Asp394 in Glt Ph changes the transporter substrate specificity to amino acid glutamate, and also leads to dependence on K + counter-transport (Wang et al., 2013). Therefore this aspartate plays a key role in both binding the amino group of substrate and coupling to K + counter-transport. Consistently, Asp394 in Glt Ph coordinates both the amino group of the bound substrate and Tl + in the Ct site. Notably, while Tl + mimics, to some extent, Na + ions in Glt Ph and EAATs, it is a better mimic of K + ions in EAATs (Boudker et al., 2007;Tao et al., 2008).

Movement of HP1 in the inward-facing state
To examine whether a complete removal of Na + and K + ions had an effect on the structure of Glt Ph in , we soaked apo Glt Ph in crystals (typically grown in the presence of K + ) in alkali-free buffer. Interestingly, we observed a small, but reproducible structural change in several crystals examined: HP1 and TM7a that form the transport domain cytoplasmic surface moved slightly towards TM8, with the tip of HP1 detaching from that of HP2 (Figure 9, Figure 9-figure supplement 1). This movement is observed clearly in one protomer (chain B in 4P3J), in which these helices are not involved in crystal packing contacts. It is reminiscent of the isomerization of the structurally symmetric HP2 and TM8a on the extracellular side of the domain observed upon the transition from bound to apo forms ( Figure 9B). It was suggested previously that HP1 participates in intracellular gating in Glt Ph (Reyes et al., 2009;DeChancie et al., 2010). Indeed, the observed movement of HP1 generates a small opening, leading to the substrate and Ct sites (Figure 9-figure supplement 2), and it is reminiscent of the movement observed in molecular dynamics simulations (DeChancie et al., 2010;Zomot and Bahar, 2013). However, this conformational difference is too small to be interpreted unambiguously.

Discussion
Our apo and ions-only bound structures reveal a remarkable structural plasticity of Glt Ph transport domain that is likely a conserved feature in the glutamate transporter family. In addition to the large trans-membrane rigid-body movements of the transport domain between outward-and inward-facing orientations, local conformational changes within the domain accompany binding and release of the transported substrate and ions ( Figure 10). These local changes provide a structural explanation of how Na + gradients are harnessed to drive concentrative substrate uptake, supporting two previously proposed coupling mechanisms (Focke et al., 2011;Reyes et al., 2013): allosterically coupled binding of the substrate and symported Na + ions, and opening of HP2 upon Na + binding, which impedes the inward trans-membrane movement of the Na + -only bound transport domain.
The apo transport domains in the outward-and inward-facing states are essentially identical and as compact as when they are fully bound, consistent with previous spectroscopic experiments (Focke et al., 2011). Therefore, the apo transport domain is likely able to transition readily between the cytoplasmic and extracellular orientations. Consistently, previous spectroscopic studies showed that the transport domains continuously sample the outward-and inward-facing positions with nearly equal probabilities either when bound to Na + and L-asp or when free of the solutes (Akyuz et al., 2013;Erkens et al., 2013;Georgieva et al., 2013;Hanelt et al., 2013). Moreover, these transitions are more frequent in the apo transporter, consistent with a lack of large energetic barriers (Akyuz et al., 2013). In Glt Ph , the compact translocation-competent apo conformation of the transport domain is stabilized by interactions between the collapsed HP2, and HP1, TM7, and TM8. In EAATs, by contrast, we speculate that these interactions are insufficient and that K + binding to the Ct site is required to and L-asp aspartate and Na + ions bound to the Na1 and Na2 sites in the fully bound transport domain (right). stabilize the translocation-competent closed conformation that can return to the outside, ensuring coupling between substrate uptake and counter-transport of K + ion. Local structural differences in EAATs in the vicinity of the Ct site may underlie the higher affinity and specificity of this site for K + ion.
In conclusion, we have shown structurally that ion binding and unbinding events in Glt Ph and, by analogy, in EAATs control the conformational state of the transporter, determining its competence to bind substrate and undergo transitions between the outward-and inward-facing states. Studies establishing the location of the Na3 binding site; the potential role of the Ct site in binding K + ; and the gating mechanism in the inward-facing state will be necessary to verify and refine our proposed mechanisms.

Materials and methods
DNA constructs, mutagenesis, protein expression, and purification R397A mutation was introduced by PCR into Glt Ph containing seven point mutations to histidine (Yernool et al., 2004), referred as wild type Glt Ph for brevity. Proteins were produced in Escherichia coli DH10b strain (Invitrogen, Inc., Grand Island, NY) as fusions with a thrombin cleavage site, and a metalaffinity octa-histidine at their carboxyl-terminus. Proteins were purified by nickel-affinity chromatography, digested with thrombin to remove the affinity tag, and purified by size exclusion chromatography (SEC) in appropriate buffers as described below. Protein concentrations were determined by measuring the absorbance at 280 nm using an extinction coefficient of 26,820 M −1 .cm −1 .

Crystallization, and soaking experiments Outward-facing state
Glt Ph -R397A was purified by SEC in 10 mM HEPES/Tris, pH 7.4, 200 mM choline chloride and 7 mM n-decyl-α-D-maltopyranoside (Anatrace, inc., Maumee, OH). Crystallization experiments were setup using the hanging-drop vapor diffusion method, by mixing protein (∼7 mg/ml) and well solutions (1:1 vol:vol), and incubated at 4°C. Na + -bound Glt Ph -R397A crystals were grown in 18-20% PEG 400, 0.1 M citric acid/Tris pH 4.5 and 0.4 M NaCl. Fully bound Glt Ph -R397A crystals were obtained in the same crystallization conditions supplemented with 5 mM L-asp. The crystals were cryo-protected by soaking in the well solution supplemented with 10% glycerol and 7 mM n-decyl-α-D-maltopyranoside and frozen in liquid nitrogen. Apo crystals were grown in 18-20% PEG 400, 0.1 M citric acid/Tris pH 4.5, and 0.4 M choline chloride. Tl + -bound Glt Ph -R397A crystals were obtained by soaking Glt Ph -R397A apo crystals in several changes of solutions containing 18-20% PEG 400, 0.1 M citric acid/Tris pH 4.5, 0.1 M TlNO 3 , 7 mM n-decyl-α-D-maltopyranoside and 10% glycerol. Figure 10. Proposed transport cycle for Glt Ph and EAATs. Ion binding to the Na1 site of the outward-facing apo transport domain triggers isomerization into bound-like conformation, formation of the L-asp and Na2 binding sites and HP2 opening, impeding translocation of the domain. Closure of HP2, coupled to L-asp and Na2 binding, allows translocation. After the release of the ligands into the cytoplasm by as yet an unknown gating mechanism, the domain is in a compact apo state, and returns to the extracellular side. Notably, binding of cations to the inward-facing state does not lead to a crystallographically observed gate opening that would impede translocation. However, Na + affinity in this state is only ∼250 mM (Reyes et al., 2013), and it will remain largely unbound when facing the cytoplasm. Hence, uncoupled Na + transport should be limited. In EAATs, an open conformation of the gates might be more favored in the apo state, and K + binding at the Ct site might be required to stabilize translocation-competent conformation of the apo transport domain. DOI: 10.7554/eLife.02283.027

Inward-facing state
Glt Ph -K55C-A364C was purified by SEC in 10 mM HEPES/KOH, pH 7.4, 150 mM KCl, and 7 mM n-decyl-α-D-maltopyranoside. Cross-linking was carried out by mixing protein at ∼3 mg/ml with a 10-fold molar excess of HgCl 2 for 20 min on ice. The samples were diluted ∼10-fold with the SEC buffer and concentrated to remove partially the excess of HgCl 2 . For crystallization, the protein samples at 2.75 mg/ml were supplemented with 20-50 mM MgCl 2 and 0.2 to 0.5 mM of a mixture of E. coli polar lipid extract and egg PC (3:1 wt:wt) (Avanti Polar Lipids, Inc., Alabaster, AL) and incubated on ice for 45 min. Crystallization was carried out using hanging drop diffusion methods in 96-well plates at 4°C. Initial crystallization conditions were identified using a replica of the crystallization screen MemGold (Molecular Dimensions., Altamonte Springs, FL), in which Na + -containing compounds were replaced with K + -containing compounds. The screen was prepared using the liquid handler Formulator (Formulatrix, Inc., Waltham, MA). Glt Ph in crystals were grown in 14-20% PEG 400, and 0.1 M potassium citrate, pH 5.0 to 6.0. For Tl + soaking, crystals were washed in 20% PEG 400, 20 mM MES/Tris, pH 6.5, 20 mM KNO 3, 20 mM MgNO 3 , and 10 mM n-decyl-α-D-maltopyranoside, and then incubated in 20% PEG 400, 20 mM MES/Tris, pH 6.5, 5 mM MgNO 3 , 10 mM n-decyl-α-D-maltopyranoside, and 150 mM TlNO 3 . In ion competition experiments, the soaking solution was further supplemented with either NaNO 3 or KNO 3 . To obtain an alkalifree structure, crystals were soaked in solution containing 20% PEG 400, 20 mM MES/Tris, pH 6.5, 5 mM MgCl 2 , and 10 mM n-decyl-α-D-maltopyranoside. Crystals were directly frozen in liquid nitrogen.

Data collection and structure determination
Diffraction data were collected at the National Synchrotron Light Source beamlines X25 and X29 (Brookhaven National Laboratory). Data from crystals soaked in Tl + were collected at a wavelength of 0.97 Å. Data were processed using HKL2000 (Otwinowski and Minor, 1997), and further analyzed using the CCP4 program suite (Collaborative Computational Project, 1994). Anisotropy correction was performed as described previously (Strong et al., 2006). Briefly, resolution limits along the a, b, and c axes were determined using the UCLA-MBI Diffraction Anisotropy server (http://services.mbi. ucla.edu/anisoscale/) and applied as cutoffs to truncate the dataset obtained after processing of diffraction images. After scaling in HKL2000, structure factors were anisotropically scaled using PHASER (McCoy et al., 2007), and a negative B factor correction was applied to these structure factors using CAD. Initial phases were determined by molecular replacement with PHASER (McCoy et al., 2007), using the structure of Glt Ph either in the outward-facing state (PDB code 2NWX) or the inward-facing state (PDB code 3KBC) as the search model. Refinement was carried out by rounds of manual model building in COOT (Emsley and Cowtan, 2004) and refinement in REFMAC5 with TLS (Winn et al., 2001). With the exception of the analysis of the data from alkali-free Glt Ph in crystals, where protomers in the trimer were clearly not identical, the electron density maps and the anomalous difference Fourier maps were three or sixfold averaged in real space. Strict non-crystallographic symmetry constrains were also applied during structural refinement in REFMAC5 when necessary. Structures of the transport domain were superimposed and R.M.S.D.s calculated using VMD software (Humphrey et al., 1996). All structural figures were prepared using Pymol (DeLano Scientific, LLC) (DeLano, 2008).

Isothermal titration calorimetry (ITC)
ITC experiments were performed as described previously (Reyes et al., 2013). Briefly, Glt Ph mutant proteins were purified by SEC in 10 mM HEPES/Tris, pH 7.4, 200 mM choline chloride, 0.5 mM n-dodecyl-β-D-maltopyranoside and concentrated to 4 mg/ml. The protein was diluted to 40 μM in buffer containing 20 mM HEPES/Tris, pH 7.4, 200 mM choline chloride, 1 mM n-dodecyl-β-D-maltopyranoside and various NaCl concentrations. ITC experiments were performed using a small cell NANO ITC (TA instruments, Inc., New Castle, DE) at 25°C. Protein samples were placed into the instrument cell and titrated with L-asp solution prepared in the same buffer. The isotherms were analyzed using the NanoAnalyze software (TA instruments, Inc., New Castle, DE), and fitted to independent binding sites model.

Fluorescence-based binding assays
Fluorescence-based binding assays were performed as described previously (Reyes et al., 2013). In brief, 100 μg/ml of protein in 20 mM HEPES/Tris, pH 7.4, 200 mM choline chloride, 0.4 mM n-dodecylβ-D-maltopyranoside, 200 nM styryl fluorescent dye RH421 (Invitrogen, Inc., Grand Island, NY) were titrated with L-asp in the presence of various concentrations of NaCl at 25°C. Fluorescence experiments were carried out using a QuantaMaster (Photon International Technology, Inc., Edison, NJ). The RH421 dye was excited at 532 nm, and the fluorescence was collected at 628 nm. Fluorescence emissions were measured after at least 1000 s equilibration. The data were analyzed using SigmaPlot12 (Systat software, Inc., San Jose, CA). Fractional fluorescence changes were corrected and normalized with respect to the dilution factors and maximal fluorescence changes, respectively. Corrected fluorescence changes were plotted as a function of ligand concentration and fitted to the Hill equation. Sodium activity was calculated as γ × [Na + ], where γ is the activity coefficient. The activity coefficient is calculated with the Debye-Hückel equation as described (Reyes et al., 2013). All the experiments were performed at least in triplicate.

Sequence analysis
Sodium:dicarboxylate symporter family sequences were harvested from PFAM database (PF00375) (Finn et al., 2008), parsed to remove incomplete sequences and sequences with over 70% identity and aligned in ClustalW (Larkin et al., 2007). The alignment was manually adjusted and the final dataset containing 463 aligned sequences was used to generate a consensus sequence using WebLogo (Crooks et al., 2004).

Molecular modeling
To model the structures of the transport domains with HP2 in the bound conformation and the NMD motif in the apo conformation, and vice versa, we superimposed the structures of the fully bound and apo forms of the transport domains using TM6, HP1, and TM7. We then generated new coordinates files combining the coordinates of TM7, including the NMD motif, from the bound form and the coordinates for HP2 from the apo form or vice versa. In both of these models, we observed steric clashes between Met311 and residues in HP2. To construct a model of the intermediate state with an open tip of HP2, we superimposed the structure of Na + -only bound Glt Ph -R397A and the intermediate state (PDB accession code 3V8G) using TM6, HP1, and TM7. We then replaced HP2 in the structure of the intermediate with HP2 from Na + -only bound Glt Ph -R397A. We moved slightly the side chain of Lys55 that was involved in a minor steric clash with the HP2 tip. We observed no major steric clashes in the resulting model.