Large domain movements through lipid bilayer mediate substrate release and inhibition of glutamate transporters

Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homologue, sodium and aspartate symporter GltPh. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of GltPh reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer.

state (IFS), from which the substrate is released into the cytoplasm, involves a rigid-body "elevator-like" movement of the transport domain by ca 15 Å across the lipid membrane (Reyes et al., 2009;Akyuz et al., 2013;Erkens et al., 2013;Ruan et al., 2017). The structures of the apo transporters in the OFS and IFS showed similar positions of the transport domains that have undergone local structural rearrangements associated with the release of the bound L-asp and Na + ions (Jensen et al., 2013;Verdon et al., 2014).
The OFS and IFS conformations show a remarkable internal symmetry (Yernool et al., 2004;Crisman et al., 2009;Reyes et al., 2009). In particular, the transport domains feature two pseudo-symmetric helical hairpins (HP) 1 and 2. HP1 lines the interface between the transport and scaffold domains in the OFS, reaching from the cytoplasmic side of the transporter. HP2 lies on the surface of a large extracellular bowl formed by the transporter and occludes L-asp and three Na + -binding sites (NA1, 2, and 3). The two hairpins meet near the middle of the lipid bilayer, and their non-helical tips provide essential coordinating moieties for the bound L-asp. As the transport domain translocates into the IFS, HP2 replaces HP1 on the domains interface, while HP1 now lines an intracellular vestibule leading to the substrate-binding site (Figure 1 Supplementary Figure 1). Structural and biophysical studies have established that HP2 serves as the extracellular gate of the transporter (Boudker et al., 2007;Focke et al., 2011;Verdon et al., 2014;Riederer e Valiyaveetil, 2019). HP2 closes when the transporter is bound to Na + ions and L-asp and when it is empty (Yernool et al., 2004;Jensen et al., 2013;Verdon et al., 2014). In contrast, it assumes open conformations when the transporter is bound only to Na + ions or Na + ions and competitive blockers DL-threo-β-benzyloxyaspartate (TBOA) or (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) (Boudker et al., 2007;Verdon et al., 2014;Canul-Tec et al., 2017).
The gating process in the IFS is less well understood. Based on symmetry considerations, it was first proposed that HP1 might serve as the intracellular gate (Yernool et al., 2004) or that the very tip of HP2 might open to release the substrate and ions (Dechancie et al., 2011). A large opening of HP2 seemed unlikely because of the steric constraints on the domain interface. However, later structures of a gain-of-function mutant of GltPh and human homologous neutral amino acid transporter ASCT2 showed that the transport domain in the IFS could swing away from the scaffold, opening a crevice between the domains (Akyuz et al., 2013;Garaeva et al., 2018). In this so-called "unlocked" conformation, there was sufficient space for HP2 to open. More recent studies of ASCT2 and of an archaeal GltTk further showed that HP2 could open, suggesting that it serves as a gate in both the OFS and IFS (Garaeva et al., 2019;Arkhipova et al., 2020). Here, we report a series of Cryo-EM structures of GltPh reconstituted into nanodiscs in the IFS and OFS. We show that the transport domain explores a large range of motions in the IFS to which the bilayer adapts through significant bending. These motions are coupled to local changes in HP2 to mediate variable substrate-binding sites exposures to the solvent and accommodate ligands of diverse sizes. They also affect the area of the hydrophobic interface between the transport and scaffold domains. When the transporter is bound to non-transportable blockers or Na + ions only, the area is significantly larger than when the transporter is apo or fully loaded with the substrate and ions. The more extensive interface may contribute to the inability of the transport domains to return to the OFS, providing a mechanism of inhibition and coupled transport.

Large range of motions of the transport domain in the IFS
In the outward-facing GltPh and EAAT1 in complex with blockers TBOA and TFB-TBOA or Na + ions only, HP2 opens to various degrees, enabling access to the substrate-binding site (Boudker et al., 2007;Verdon et al., 2014;Canul-Tec et al., 2017). To picture gating in the IFS, we imaged the GltPh reconstituted into MSP1E3 nanodiscs in the presence of various ligands by single-particle Cryo-EM. We used a variant of GltPh, conformationally constrained in the IFS by cross-linking of cysteine residues placed into the transport and scaffold domains, GltPh-K55C/A364C (GltPh IFS ) (Reyes et al., 2009). Earlier crystal structures of GltPh IFS pictured the position of the transport domain that was very similar to those visualized in unconstrained inward-facing GltPh mutants (Verdon e Boudker, 2012;Akyuz et al., 2015).
We determined the structures of GltPh IFS free of ligands (GltPh IFS -Apo-open) or in complex with Na + ions (GltPh IFS -Na) and bound to L-asp (GltPh IFS -Asp), TBOA (GltPh IFS -TBOA),   TFB-TBOA (GltPh IFS -TFB-TBOA), and the wide-type outward-facing GltPh in complex with TBOA (GltPh OFS -TBOA) to 3.52, 3.66, 3.05, 3.66, 3.71, and 3.66 Å resolution, respectively (Methods, Figure 1 Supplementary Figures 2-4, and Table 1). The GltPh IFS -Asp structure was nearly identical to the earlier crystal structure (Reyes et al., 2009). The transport domain was well packed against the scaffold primarily through interactions of HP2 and the extracellular part of TM8 (TM8a) with the scaffold TMs 2, 4, and 5. The central axis of the roughly cylindrical transport domain formed a ~35 ° angle Figure 1. Gating mechanism in the inward-facing state. a, Structures of GltPh protomers are shown in surface representation viewed in the membrane plain. The scaffold domain is colored wheat, the transport domain blue, HP1 yellow, and HP2 red. The PDB accession code for GltPh IFS -Apo-closed is 4P19. An approximate position of the bilayer is shown as a pale orange rectangle. b, Angles between the membrane normal drawn through the center of the scaffold domain and the central axis of the transport domains are shown for GltPh IFS -Apo-closed and GltPh IFS -TFB-TBOA (top). Distances between the cα atoms (black circles) of residues R276 and P356 are shown for the same structures under the schematic depiction of the transport domains. Corresponding angles and distances are listed under all structures in panel a. c, A schematic representation of the gating mechanism on the extracellular (top) and intracellular (bottom) sides of the membrane.
with the membrane normal (Figure 1a, b). HP2 was closed over the substrate-binding site, and packing between the transport and scaffold domain left no space for the hairpin to open.
A similar inter-domain orientation and packing were also observed in a crystal structure of the occluded apo GltPh IFS (GltPh IFS -Apo-closed, PDB code 4P19, Figure.1a). In the new structures of GltPh IFS -Na, GltPh IFS -Apo-open, GltPh IFS -TBOA, and GltPh IFS -TFB-TBOA, approximately the same regions of HP2 and TM8a remained juxtaposed against the scaffold. However, the bulk of the transport domain swung out away from HP2 and the scaffold to different extents (Figure 1a) with the largest angle between the transport domain and the membrane normal of ~47° in GltPh IFS -TFB-TBOA (Figure 1b). Together, the crystal and Cryo-EM structures define mechanisms of gating in GltPh on the extracellular and cytoplasmic sides of the membrane (Figure 1c, Movie 1). In the OFS, the bulk of the transport domain remains mostly static relative to the scaffold, and the labile HP2 serves as the extracellular gate. In the IFS, HP2 can maintain interactions with the scaffold, while the bulk of the transport domain swings away to allow access to the binding site. Notably, in a crystal structure of a gain-of-function L-asp-bound mutant GltPh IFS -R276S/M395R, the transport domain is positioned at ~45 ° angle, similar to the GltPh IFS -TFB-TBOA (Akyuz et al., 2015). However, HP2 remains closed over the binding site and a large lipid-filled gap forms between the transport and scaffold domains. It is currently unclear whether the transport domain first swings away from the scaffold providing space for the consequent HP2 opening or whether HP2 remains in place while the bulk of the domain swings out in a "wag-the-dog" manner.

Two transporter blockers bind differently to GltPh IFS
TBOA and TFB-TBOA blockers share the amino acid backbone with L-asp but are decorated on β-carbon with one and two bulky benzyl rings, respectively, that cannot fit within the confines of the substrate-binding site. They block transport by binding to the outward-facing GltPh or EAATs and arresting HP2 in an open conformation (Boudker et al., 2007;Canul-Tec et al., 2017). Our Cryo-EM structure of the outward-facing GltPh OFS -TBOA in nanodisc confirmed that the transporter took the same conformation in the absence of crystal contacts in a lipid bilayer (Figure 2 Supplementary Figure 1a).
Because TBOA also binds to the IFS of GltPh (Reyes et al., 2013;Oh e Boudker, 2018), we determined the structures of the GltPh IFS complexes with the blockers. In the GltPh IFS -TBOA structure, the ligand density was resolved for the aspartate moiety. However, the density for the benzyl group was lacking, perhaps because the blocker is a mixture of L and D isomers leading to heterogeneity (Figure 2 Supplementary Figure 1b).
In contrast, in the GltPh IFS -TFB-TBOA structure, TFB-TBOA density was clear, and we modeled the inhibitor in its binding site (Figure 2a). We also modeled L-asp into the excess density in the binding site of GltPh IFS -Asp (Figure 2 Supplementary Figure 1c).
The bound L-asp and TFB-TBOA share some critical interactions (Figure 2b). Thus, R397 coordinates the side chain carboxylates of aspartate moieties, and D394 coordinates the amino groups. However, TFB-TBOA assumes a different rotomer, leading to the The corresponding density is shown as a black mesh object. Red arrow emphasizes HP2 opining. b, TFB-TBOA assumes a distinct rotomer and is coordinated differently from L-asp. c, Superimposed GltPh IFS transport domains in complex with L-asp (grey) and TBOA (colored). Red arrow emphasized parting of the N-and C-terminal arms of HP2. L-asp and Na + ions are shown as spheres. d, Two mechanisms of blockers binding to GltPh IFS showing either opening of HP2 or parting of the two arms to accommodate the bulky moieties of the blockers.  Figure 1d). Interestingly, HP2 was in the same conformation also in an R397C GltPh mutant bound to glutamine or benzyl-cysteine. In these structures, the ligands made virtually no interactions with the hairpin but introduced steric clashes disallowing the loop closure (Scopelliti et al., 2018). Therefore, it appears that the hairpin intrinsically favors this open conformation.
Surprisingly, HP2 does not open in the same way in GltPh IFS -TBOA. Instead, the hairpin remains mostly closed and its N-and C-terminal arms part, perhaps to provide space for the benzyl group (Figure 2c). This movement disrupts the Na2 binding site, consistent with previous observations that binding of TBOA and related L-β-threo-benzyl-aspartate to the IFS of the transporter required only two Na + ions (Reyes et al., 2013). The movement creates a small opening into the cytoplasmic milieu between the tips of HP1 and HP2. It is not clear whether this conformation reflects a functional state. Perhaps, it recapitulates a transient transporter state, in which a Na + ion has already left the Na2 site while the substrate and two remaining Na + ions are still bound. Water might use the cytoplasmic opening to reach and eventually displace the remaining solutes.
Collectively, the structures show that in GltPh IFS , bulky competitive inhibitors can be accommodated either by opening HP2 or by parting its N-and C-terminal arms ( Figure   2d). Since the OFS and IFS share the same binding pocket for the substrate and competitive inhibitors, it is likely that the new mode of inhibitor binding, which involves parting of the HP2 arms, can be sampled in the OFS as well. The novel mode of blocker binding might provide new pharmacological avenues for the inhibition of human glutamate transporters.

M311 and R397 couple HP2 gating to ion and substrate binding.
To further explore the gating mechanism, we aimed to resolve a structure of Na + onlybound GltPh IFS and imaged nanodisc-reconstituted GltPh IFS frozen in the presence of 200 mM NaCl (Figure 1 Supplementary Figure 3b and 4, and Table 1). We isolated two distinct structural classes of GltPh IFS protomers after symmetry expansion and classification without alignment. The structural heterogeneity was not surprising in retrospect because Na + concentration in the sample was close to the dissociation constant measured for GltPh IFS (Reyes et al., 2013). Thus, we observed both Na + -bound and apo states of the transporter, GltPh IFS -Na, and GltPh IFS -Apo-open, respectively. The states were assigned based on the conformations of the conserved non-helical NMD motif (residues 310-312) in TM7, which coordinates of Na + ions in the Na1 and Na3 sites, and TM3, part of the Na3 site (Figure 3 Supplementary Figure 1a). In particular, the side chain of M311 protrudes towards the L-asp and Na2 sites in GltPh IFS -Na and GltPh IFS -Asp structures. In contrast, it flips out toward TM3 in our GltPh IFS -Apo-open structure and a published GltPh IFS -Apo-closed crystal structure (Verdon et al., 2014). We did not observe density for Na + ions in the Na1 and Na3 sites of GltPh IFS -Na. However, all ion-coordinating residues are positioned similarly to GltPh IFS -Asp (Figure 3 Supplementary Figure 1b). Notably, Na1 is coordinated in GltPh IFS -Asp, in part, by an occluded water molecule. In GltPh IFS -Na, the water is no longer occluded and is part of an aqueous cavity (Figure 3a). We conclude that ions likely occupy Na1 and Na3 sites, but the Na1 site might be in rapid equilibrium with the solution.
Surprisingly, GltPh IFS -Apo-open differs significantly from the earlier occluded crystal structure GltPh IFS -Apo-closed in that the substrate-binding site is open and hydrated. The opening resembles that in GltPh IFS -Na compared to the occluded GltPh IFS -Asp (Figure 3a, b) and shares the overall mechanism: HP2 remains in contact with the scaffold while the rest of the transport domain swings out (Figure 1c). From the viewpoint of the transport domain, the conformational changes lead to a similar HP2 opening (Figure 3c, d, Figure   3 Supplementary Figure 2a). Interestingly, in GltPh IFS -Na, there is also a small shift of HP1 away from the substrate-binding site, possibly increasing water access to the Na1 site.
A similar small movement of the otherwise rigid HP1 was observed in the crystals of apo GltPh IFS grown in an alkali-free buffer (Verdon et al., 2014).
Two residues in the transport domain -M311, and R397 -move significantly during gating and might couple solute binding and release to the large-scale conformational changes.
Here we consider a sequence of structural events, which might underlie ion and substrate release in the IFS (Figure 1c), starting with GltPh IFS -Asp and going to GltPh IFS -Na, GltPh IFS -Apo-open, and GltPh IFS -Apo-closed (Movie 2). In GltPh IFS -Asp, the R397 side chain extends upward, toward the extracellular side of the membrane, where D390 coordinates its guanidinium group. Thus positioned, R397 makes space for L-asp and coordinates its sidechain carboxylate, while D394 coordinates its amino group (Figure 3e). M311 protrudes into the binding site and coordinates Na2 (  The binding site is occluded in Na + /L-asp-bound, and closed apo (PDB 4P19) states and is exposed to the solvent in Na + -only, and apo open states. c, Superimposed transport domains of GltPh IFS -Na (colored) and GltPh IFS -Asp (grey). L-asp and Na + ions are shown as spheres. Yellow and red arrows indicate movements of HP1 and HP2, respectively. d, Superimposed transport domains of GltPh IFS -Apo-open (colored) and GltPh IFS -Apo-closed (grey). e, Gating steps in the inward-facing state. Top: Local structural changes upon L-asp and Na2 release from GltPh IFS -Asp (grey), leading to an open state of GltPh IFS -Na (colored). Black arrows indicate the release of L-asp and Na2 and associated opening of HP1 and HP2. Bottom: Binding site occlusion transition from GltPh IFS -Apo-open (colored) to GltPh IFS -Apo-closed (grey). Black arrows mark movements of R397 into the binding site and the concomitant closure of HP2.
Supplementary Figure 2b). The consequent release of Na1 and Na3 leads to a restructuring of the NMD motif and a large outward rotation of M311, which now packs against the open HP2 of GltPh IFS -Apo-open (Figure 3 Supplementary Figure 2b). The guanidinium group of R397 remains between D390 and D394. To achieve the closed apo state, M311 swigs further out into the lipid bilayer, allowing HP2 to close. R397 descends deep into the binding pocket, coordinated now only by D394, and is poised to make direct or through-water interactions with carbonyl oxygens of the closed tip of HP2. Steric hindrance of M311 and more positive local electrostatics may prevent R397 from entering the L-asp binding site, and closing HP2 in Na + -only bound GltPh IFS . Physiologically, such Na + -bound occluded states should be avoided to prevent Na + leaks.
Interestingly, in our Cryo-EM analysis, we did not find any GltPh IFS -Apo-closed structures previously visualized by crystallography. It might be that the open conformation of the apo GltPh IFS is the preferred state of the transporter and that the GltPh IFS -Apo-closed state is assumed only transiently, before the outward transition of the transport domain. Packing crystal contacts might have stabilized the closed conformation.

Ligand-dependent domain interface.
HP2 and TM8a comprise most of the transport domain surface interacting with the scaffold.
Strikingly, in each of our IFS structures, HP2 takes a different conformation (Figure 4 Supplementary Figure 1a). These are similar in structures with Na + ions bound to Na1 and Na3, i.e., in complexes with Na + ions only and with L-asp, TBOA, or TFB-TBOA.
The differences are mostly around the tips of HP2 near the L-asp and Na2 sites ( Thus, the positions of HP2 tips on the domain interface are mostly conserved. The structural differences in the hairpins then lead to their different orientations relative to the scaffold and different positions of the transport domains, which lean away from the scaffold and rotate around a membrane normal. The rotation is small for GltPh IFS -Apoclosed, relative to GltPh IFS -Asp (7°), but is significant for GltPh IFS -Na (23°), and GltPh IFS -TFB-TBOA (29°). A consequence of these differences is that the bulky residues in the HP2 N-terminal arm, L339, L343, L347, and I350 make more extensive interactions with the scaffold TMs 4a and 4c in GltPh IFS -Na and GltPh IFS -TFB-TBOA compared to other structures. Furthermore, interaction areas between HP2/TM8 and the scaffold domain differ, with GltPh IFS -Apo-closed and GltPh IFS -Asp structures having the smallest area of 1086 and 1076 Å 2 , respectively, and GltPh IFS -Na showing the largest increase of ~400 Å 2 (Figure 4).
The disruption of the interdomain interface is a prerequisite for the transport domain translocation from the inward-to the outward-facing position. Therefore, altered geometry of the interface and larger interaction area may explain why translocation is inhibited by blockers TBOA, and TFB-TBOA, or especially in the transport domain bound to Na + ions only. While it is not possible to translate interaction areas into energies, it is notable that translocation-competent closed apo and L-asp-bound states show the smallest areas.
Consistently, the gain of function mutant R276S/M395R shows a reduced interaction area of only 543 Å 2 and a translocation rate several-fold faster than the wild type transporter (Akyuz et al., 2015).

Transport domain movements coupled to lipid bilayer.
The Cryo-EM structures of the outward-and inward-facing states of GltPh are overall similar to the crystal structures. However, they differ in the N-terminus, which is We also find lipid moieties, structured to various degrees, in the crevices between the scaffold and transport domains (Figure 5a). Of these, the most notable one is inserted between the N-terminal arm of HP2 and the scaffold TM4a (LipidOut Figure 5a, b).
Interestingly, we observe lipids at almost the same location in the outward- (Huang et al., 2019) and inward-facing L-asp-bound transporters (Figure 5b). The lipid packs similarly against TM4a in the OFS and IFS but interacts differently with HP2: near the tip and the extracellular base, respectively. It is not yet clear whether during the outward-to-inward transition, as HP2 slides past TM4a, the lipid is temporarily displaced or disordered.
Interestingly HP2 opening in the OFS, as seen in GltPh OFS -TBOA, and the IFS, as seen in the current study, requires displacement of LipidOut. Thus, the lipid molecules at this site could modulate gating and the translocation dynamics, affecting both substrate affinity and transport rate. In GltPh IFS -TFB-TBOA and GltPh IFS -Apo-open structures, the transport domain leans away from the scaffold far enough to open a window between the two domains that connects the interior of the bilayer to the solvent-filled crevice on the cytoplasmic side of the transporter (Figure 5c). We observe excess densities in the opening, suggesting that lipids enter the space at a position structurally symmetric to LipidOut (LipidWindow, Figure 5c).
Perhaps most strikingly, we observe overall distortions of the nanodisc correlated to the position of the transport domain (Figure 5d, Movie 3). The nanodisc is nearly flat in the structure of the GltPh OFS -TBOA, in which the hydrophobic regions of the transport domain and the scaffold are aligned. In GltPh IFS -Asp, the transport domain is positioned at the sharpest angle to the membrane normal (Figure 1a), and its hydrophobic region descends the furthest toward the cytoplasm. The resulting hydrophobic mismatch between the scaffold and transport domains leads to membrane bending to accommodate both, as has been predicted by computational studies (Zhou et al., 2019). In other inward-facing Figure 5. Coupling of the lipid bilayer to protein motions. a, Lipid densities (red) observed in protein cervices of GltPh IFS -Asp. Lipid molecules tucked in between the N-terminus and the rest of the scaffold (Lipidin) are observed in all OFS and IFS structures. b, lipid densities (red mesh objects, Lipidout) observed on the extracellular side of a crevice between the scaffold TM4a and HP2 in both outward-(PDB code 6UWF) and inward-facing GltPh bound to L-asp. c, Density map of a GltPh IFS -TFB-TBOA protomer, with the lipid density in the window between the transport domain and scaffold colored red (Lipidwindow). d, Density map of GltPh OFS -TBOA, GltPh IFS -Asp, and GltPh IFS -TFB-TBOA in nanodiscs viewed in the membrane plain. Density corresponding to the transport and scaffold domains are colored blue and wheat, respectively. Density corresponding to the nanodisc is colored yellow. Black arrows mark deviations of the nanodiscs from the planar structures. structures, where the transport domains swing out, the hydrophobic regions are positioned further up toward the extracellular side of the membrane leading to reduced membrane bending. In the GltPh IFS -TFB-TBOA structure, the membrane bends in the reverse direction as the swing of the transport domains brings their hydrophobic regions above those of the scaffold (Figure 5d, Movie 3).

Discussion
The series of structures that we have determined by Cryo-EM suggest that both substrate Our structures show that the release of Na + ions and L-asp requires movement of the transport domain, mediated by conformational changes of HP2 and the HP2/TM8ascaffold interface. The complexity of these conformational events may explain why the substrate binding and release are slow in the IFS (Oh e Boudker, 2018), while fast in the OFS (Hanelt et al., 2015). Notably, kinetic studies showed that the release (and binding) of one Na + ion in the IFS, most likely Na2, is rapid (Oh e Boudker, 2018). Thus, it is likely that the release of Na2 requires little structural change, limited at most to the change observed in the GltPh IFS -TBOA structure. Our structural data further suggest that mutations in HP2 may increase the substrate dissociation rate in the IFS by increasing the dynamics of the hairpin and the hairpin/scaffold interface.
Single-molecules studies of the OFS to IFS translocation dynamics showed that the ratelimiting high-energy transition state most likely resembles the IFS structurally and that the transport domain might make multiple attempts to achieve a stable observable IFS (Huysmans et al., 2020). These studies suggest that multiple IFS conformations exist and are separated by significant energetic barriers. While our structures most likely represent the lowest-energy states, their multiplicity in itself supports the existence of a complex conformational ensemble sampled by the transporter in the IFS. Other lipids, in particular extracellular LipidOut, present in the OFS and IFS, and the symmetric cytoplasmic LipidWindow observed in the IFS, have to move in and out of their binding sites during the transport cycle. Interestingly, LipidOut is inserted between the Nterminal arm of HP2 and the scaffold in both the OFS and IFS. Thus, the HP2 arm shows a conserved behavior in the two states: When HP2 occludes the L-asp binding site, LipidOut fills the space between the arm and the scaffold, but when HP2 opens during gating, the arm interacts directly with the scaffold displacing the lipid. In contrast, LipidWindow, in the IFS, moves in when HP2 opens to release the substrate and moves out when HP2 closes.
Such intimate involvement of lipids suggests that they have the potential to regulate both substrate affinity and conformational dynamics of the transporter. So far, however, only modest effects of specific lipids on GltPh transport activity have been reported (Mcilwain et al., 2015). Interestingly, in mammalian EAAT1 and ASCT2, similar space between the N-terminal arm of HP2 and scaffold is observed in the OFS, and IFS (Canul-Tec et al., 2017;Guskov et al., 2018;Yu et al., 2019), and likely can accommodate lipids. These proteins are known to be regulated by lipids, particularly by the arachidonic acid (Zerangue et al., 1995;Fairman et al., 1998;Tzingounis et al., 1998), and the identified lipid-binding sites might mediate these effects.
The lipid bilayer bending to accommodate conformational change from the OFS to IFS has been observed in recent molecular dynamics simulations (Zhou et al., 2019). The computational study suggests that the energy penalty associated with bilayer bending might be as large as 6-7 kcal/mol protomer. Our results show that not only the OFS to IFS transitions, but also the substrate release in the IFS involve changes in membrane deformation. Thus, large energetic costs of membrane bending might accompany glutamate transporter functional cycle, suggesting that the physical properties of lipid bilayers, such as thickness and stiffness (Lundbaek et al., 2010;Bruno et al., 2013;Rusinova et al., 2014), might have large impacts on transporter function.

GltPh expression, purification and crosslinking
The fully functional seven-histidine mutant of GltPh that has been used in previous studies and that is referred to as wildtype (WT) for brevity, and the K55C/C321A/A364C GltPh mutant were expressed as C-terminal His8 fusions and purified as described previously (Yernool et al., 2004). Briefly, the plasmids were transformed into E. coli DH10-B cells (Invitrogen). Cells were grown in LB media supplemented with 0.2 mg/l of ampicillin (Goldbio) at 37 °C until OD600 of 1.0. Protein expression was induced by adding 0.2 % arabinose (Goldbio) for 3 hr at 37 °C. The cells were harvested by centrifugation and resuspended in 20 mM Hepes, pH 7.4, 200 mM NaCl, 1 mM L-asp, 1 mM EDTA. The suspended cells were broken using Emulsiflex C3 high pressure homogenizer (Avestin Inc.) in the presence of 0.5 mg/ml lysozyme (Goldbio) and 1 mM phenylmethanesulfonyl fluoride (PMSF, MP Biomedicals). After centrifugation for 15 min at 5000 g at 4 °C to remove the debris, membranes were pelleted by centrifugation at 125,000 g for 60 min.

Reconstitution of GltPh into nanodiscs
Membrane scaffold protein MSP1E3 was expressed and purified from E. coli and GltPh was reconstituted into lipid nanodiscs as previously described, with modifications (Ritchie et al., 2009). Briefly, E. coli polar lipid extract and egg phosphatidylcholine in chloroform (Avanti) were mixed at 3:1 (w:w) ratio and dried on rotary evaporator and under vacuum overnight. The dried lipid film was resuspended in buffer containing 20 mM Hepes/Tris, pH 7.4, 200 mM NaCl, 1 mM L-asp and 80 mM DDM by 10 freeze/thaw cycles resulting in 20 mM lipid stock. The purified GltPh protein in DDM was mixed with MSP1E3 and lipid stock at 0.75:1:50 molar ratio at the final lipid concentration of 5 mM and incubated at 21 °C for 30 min. Biobeads SM2 (Bio-Rad) were added to one third of the reaction volume and the mixture was incubated at 21 °C for 2 hr on a rotator. Biobeads were replaced and incubated at 4 °C overnight. The sample containing GltPh IFS reconstituted into the nanodiscs in the presence of 1 mM L-asp was cleared by centrifugation at 100,000 g and purified by SEC using a Superose 6 Increase 10/300 GL column (GE Lifesciences) pre-equilibrated with buffer containing 20 mM Hepes/Tris, pH 7.4, 200 mM NaCl and 1 mM L-asp. The peak fractions corresponding to GltPh IFS -containing nanodiscs were collected for Cryo-EM imaging. To prepare substrate-free WT GltPh and GltPh IFS in nanodiscs, the reconstitution mixtures were cleared by centrifugation at 100,000 g, diluted with 10 x volume of buffer containing 20 mM Hepes/Tris, pH 7.4, and 50 mM choline chloride, and concentrated using 100 kDa cutoff concentrator. After repeating the procedure twice, substrate-free transporters in nanodiscs were purified by SEC in the same buffer. The peak fractions were collected and immediately supplemented with buffers containing 200 mM NaCl and 10 mM DL-TBOA, 200 mM NaCl and 10 mM TFB-TBOA, or 200 mM NaCl. The presence of the MSP1E3 and GltPh proteins in the samples was confirmed by SDS-PAGE. Negative staining electron microscopy was used to confirm the formation and the homogeneity of the nanodisc samples.

Image processing
The frame stacks were motion corrected using MotionCorr2 (Zheng et al., 2017) and contrast transfer function (CTF) estimation was performed using CTFFIND4 (Rohou e Grigorieff, 2015). All further processing steps were done using RELION 3.0 unless otherwise indicated (Zivanov et al., 2018). Dogpicker (Voss et al., 2009) as part of the Appion processing package (Lander et al., 2009) was used for reference-free particle picking. Picked particles were then extracted and subjected to 2D classification to generate 2D class-averages which were used as templates for automated particle picking in Relion.
The particles were extracted using a box size of 275 Å with 2x binning and subjected to 2 rounds of 2D classification ignoring CTFs until the first peak.
For GltPh IFS -Asp, GltPh IFS -TBOA, GltPh IFS -TFB-TBOA, and for the GltPh OFS -TBOA, particles selected from 2D classification were re-extracted without binning and further classified into 6 classes without enforcing symmetry using initial models generated in CryoSPARC (Punjani et al., 2017) and filtered to 40 Å. Particles from the best classes showing trimeric transporter arrangements were subjected to 3D refinement applying C3 symmetry. After conversion, the refinement was continued with a mask excluding the nanodisc. To further improve the resolution of the maps, the particles after 3D refinement were subject to an additional round of 3D classification without alignment with C3 symmetry and T=4 applying a mask to exclude the nanodisc. Particles from the best class were subjected to further masked refinement and CTF refinement. A masked refinement following CTF refinement yielded final maps with the following resolution: 3.05 Å (GltPh IFS -Asp), 3.71 Å (GltPh IFS -TFB-TBOA), 3.66 Å (GltPh IFS -TBOA), 3.66 Å (GltPh -TBOA). The resolution limits of the refined maps were assessed using Relion postprocessing and gold standard FSC value 0.143 using masks that excluded the nanodiscs.
To test for potential conformational heterogeneity, we re-processed the datasets with no symmetry applied (C1). The obtained maps showed slightly lower resolution but no detectable difference when compared to the results from the C3 refinement. We also processed the datasets with symmetry expansion (C3) and did not find additional conformations.
During processing of the data for GltPh IFS -NaCl, 529,155 particles selected from 2D classification were re-extracted without binning and were subjected to 3D classification with K=1 and no symmetry applied, using GltPh IFS -Asp map as the initial model. The same particles were subject to 3D refinement with C3 symmetry. After conversion, the refinement was continued with a mask to exclude the nanodisc, resulting in a 3.56 Å resolution map. To probe for conformational heterogeneity, we performed symmetry expansion implemented in Relion (Scheres, 2016). 1,587,465 protein subunits were rotated to the same position and subjected to a focused 3D classification without alignment with T=40 into 10 classes. The local mask was generated using Chain A of PDB model 3KBC and included only densities from one subunit of the refence map. Two different conformations were observed. From the 10 classes, 5 classes showed a conformation identified as GltPh IFS -Na and 5 classes showed a different conformation identified as GltPh IFS -Apo-open. The best GltPh IFS -Na + class (191,349 particles), which contained 12 % of the symmetry expanded protomers and the best GltPh IFS -Apo-open class (148,582 particles), which contained 9 % of the symmetry expanded particles, were separately subjected to a final focused 3D refinement with C1 using a mask to exclude the nanodisc.
The local angular searches in this refinement were conducted only around the expanded set of orientations to prevent contributions from the neighbor subunits in the same particle.
The resulting maps were postprocessed in Relion using the same mask as in 3D classification after symmetry expansion. The final resolution at gold standard FSC value 0.143 was estimated as 3.52 Å for the GltPh IFS -Apo-open map and 3.66 Å for GltPh IFS -Na map. Local resolution variations were estimated using ResMap (Kucukelbir et al., 2014).
After symmetry expansion with C3, we also tried to first subtract the density outside of one GltPh subunit and then perform 3D classification without alignment on the subtracted particles. The signal subtraction did not further improve the 3D classification and the 3D refinement.

Model building and refinement
For atomic model building from GltPh IFS -Asp, GltPh IFS -TBOA, and GltPh IFS -TFB-TBOA maps, crystal structure of GltPh in the IFS (accession code 3KBC) was docked into the density maps using UCSF Chimera (Pettersen et al., 2004). For the WT GltPh OFS -TBOA, crystal structure of GltPh in the OFS (accession code 2NWW) was docked into the density.
For GltPh IFS -Na or GltPh IFS -Apo-open, one subunit of 3KBC was docked into the density.
After the first rounds of the real-space refinement using Phenix (Afonine et al., 2010), miss-aligned regions were manually rebuilt and missing side chains and residues were added in COOT (Emsley et al., 2010). 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE) was used as a model lipid and placed into the excess densities which resembled lipid molecules. The acyl chains or ethanolamine heads were truncated to fit the visible densities. Models were iteratively refined applying secondary structure restraints and validated using Molprobity (Chen et al., 2010). For further cross validation and to check for overfitting, all atoms of each model were randomly displaced by 0.3 Å and each resulting model was refined against the first half-map obtained from processing. FSC between the refined models and the half-maps used during the refinement were calculated and compared to the FSC between the refined models and the other half-maps. In addition, the FSC between the refined model and sum of both half-maps was calculated. The resulting FSC curves were similar showing no evidence of overfitting. Defocus range (μm) -1.5 to -2.5 -1.5 to -2.5 -1.5 to -2.5 -1.5 to -2.5 -1.5 to -2.5 -1.5 to -2.5 Pixel size (Å)  Supplementary Figure 1. Schematic representation of the elevator mechanism of transport by GltPh. The scaffold domain is in wheat, and the transport domain is in gray. HP1 and HP2 are yellow and red, respectively. Substrate L-asp is represented as a letter A and the benzyl group of the blocker TBOA is shown as a green hexagon. Three symported Na + ions are shown as purple circles.