Cryo-EM structures of Glt Tk in nanodiscs

Purified Glt Tk was reconstituted into nanodiscs using the MSP2N2 scaffold protein31 and a mixture of E.coli polar lipids and egg PC (3:1 (w/w)), a lipid composition that supports robust transport activity of the protein in proteoliposomes11,32. Glt Tk -nanodiscs were concentrated to 4.5–9.0 µM, and supplemented with 300 mM Na+. To these preparations we added either nothing (Na+-only condition), or different concentrations of l-aspartate, or the competitive inhibitor dl-threo-beta-benzyloxyaspartate (TBOA inhibited). The preparations were analyzed by single particle cryo-electron microscopy with the aim to obtain structural insight in the conformational ensemble under turnover and stalled conditions (See Table 1 and Supplementary Fig. 1 for the cryo-EM workflows). We solved five structures of Glt Tk (with resolutions of 3.2–3.5 Å, Supplementary Fig. 2), each with the protomers in a different trimeric arrangement (Fig. 1). In the collective set of structures, the 15 individual protomers adopted four different conformations (Fig. 1), which are characterized by their position relative to the scaffold domain (inward, outward, or intermediate-outward), the accessibility of the aspartate binding site (open or occluded), and the presence of substrates (apo, Na+-only, holo (Asp), holo (TBOA)). With one exception (when the Glt Tk binding sites were saturated with L-aspartate), the arrangements of the transport domains in the trimer are non-symmetrical.

Table 1 Cryo-EM data collection, refinement and validation. Full size table

Fig. 1: Conformational states of the trimeric Glt Tk . a, b Volume and cartoon representation of five cryo-EM structures of Glt Tk in MSP2N2 nanodiscs obtained in the absence of substrate (Na+-only), in presence of L-aspartate (unsaturated 2in:1out, unsaturated 1in:2out, saturated) or in the presence of DL-TBOA. The approximate position of the lipid bilayer is represented by the light gray bar, with indication of sides of the membrane (in and out). a The cryo-EM density for the MSP2N2 belt is shown in dark gray. The transport domains of the individual protomers are present in four different conformations: inward open (steel blue), intermediate-outward occluded apo (cyan), intermediate-outward occluded Asp (cornflower blue), outward-open TBOA (dark blue). The scaffold domains are shown in yellow. c Table summarizing conformations of Glt Tk protomers. Schematic representation of the conformations on the right in the same colors as in a, b, with indication of l-aspartate (black circle), DL-TBOA (black square) and HP2 (dark red stick). Full size image

Aspartate-free conditions

Cryo-EM analysis of Glt Tk in the presence of 300 mM Na+, but in the absence of the substrate l-aspartate showed an asymmetric trimer with two transport domains in an inward position, and the third one in an intermediate-outward position. The latter transport domain has an occluded binding site, with the HP2 gate closed (distance between the tips of HP1 and HP2 ~ 4.4 Å, Supplementary Table 1). We infer that this protomer is in the apo state, because the conformation of the transport domain is identical to that of the transport domains in the apo-crystal structure of Glt Tk (PDB 5DWY, rmsd 0.534 Å)12. In the apo-state the binding sites of sodium ions are deformed, and the binding site for aspartate is non-existent because the conformation of the chain of Arg401 is incompatible with aspartate binding (Fig. 2a). Despite the similarity in conformation of the apo-transport domains in the cryo-EM and crystal structures, the position of the transport domain relative to the scaffold domain is different. In the cryo-EM structure, the transport domain is in an intermediate-outward position, whereas in the crystal structure (PDB 5DWY)12 it is fully outward. The position of the transport domain is roughly similar to that of an intermediate-outward domain observed in a crystal structure of Glt Ph (PDB 3V8G, chain C29; rmsd 1.261 Å, Supplementary Fig. S3a), but whereas the Glt Ph protomer was in a holo state (Na+-bound and Asp-bound), the Glt Tk protomer is in the apo state.

Fig. 2: Glt Tk substrate binding site and HP2 opening. a Overlay of apo (cyan), holo (Asp) (cornflower blue) and Na+-only states (light blue). The shift of Met314 and Arg401 is shown with arrows. b Cryo-EM density of l-aspartate (black mesh at 5σ). c Absence of the substrate in the inward open state (density is shown as gray mesh at 5σ). d Cryo-EM density of L-TBOA (black mesh at 4σ). e Slice through of TBOA-inhibited Glt Tk structure (surface representation) showing an inward-oriented and outward-oriented protomer. Opening of HP2 on both sides of the membrane prevents movements of the transport domains. f Superposition (on HP1) of the transport domains in inward Na+-only, intermediate-outward holo-Asp and fully-outward TBOA-inhibited (dark blue) states. Opening of HP2 in the inward state (4.4 Å) and TBOA-inhibited state (10.4 Å) in comparison with the occluded state is measured using Cα of Val358 (shown as sphere). l-aspartate (black sticks) indicates position of the substrate-binding site. Full size image

In the two inward-oriented transport domains the gate formed by HP2 is open (distance between the tips of HP1 and HP2 ~ 9.3 Å, Supplementary Table 1), and the empty binding site for aspartate (Fig. 2c) is accessible to the aqueous solution. The conformation of the protomers strongly suggests that the inward-open protomers are in a Na+-bound state. This interpretation is based on the observation that two of the three binding sites for Na+ ions (Na1 and Na3), which are deformed in the apo protein12, are reshaped in the cryo-EM structure, consistent with bound Na+ ions. The presence of cryo-EM density in these sites further supports the interpretation that sodium ions are indeed bound, although the densities do not allow unequivocal assignment at the obtained resolution of 3.2 Å (Supplementary Fig. 4a). The Na2 site is not formed properly, because the open HP2 gate is incompatible with the Na2 site geometry (Supplementary Fig. 4b). A prominent indicator for the Na+-bound geometry at sites Na1 and Na3 is the conformation of the central unwound region of transmembrane helix 7 (TM7), around Met314. The sidechain of Met314 points away from the binding site in the apo state, as first shown in a crystal structure of Glt Tk 12, and later also observed for Glt Ph 33 (see also Fig. 2a), but is rotated over a distance of 9.7 Å in the bound state.

Na+ binding was shown previously to lead allosterically to formation of a high-affinity aspartate-binding site11,12,34,35. In the cryo-EM structure of the inward-oriented protomers, the side chain of Arg401, which adopts a conformation incompatible with aspartate binding in the apo-state, indeed has taken the position required for high-affinity L-aspartate binding (Fig. 2a). While an l-aspartate binding site is present in these protomers, it is unoccupied, consistent with a lack of cryo-EM density (Fig. 2c). The protomers are thus in a hitherto elusive Na+-only state.

The fact that we observe one protomer in the apo state and two in Na+-bound states, indicates that we did not manage to saturate all protomers with Na+ using a concentration of 300 mM Na+. This observation is consistent with reported K d values for sodium binding to Glt Ph (100–140 mM)36. Because of the sub-saturating Na+ concentration, Glt Tk trimers with different ratios of apo and Na+-bound protomers were also expected in the dataset. Analysis of particles discarded during heterogeneous refinement for this dataset indeed revealed the presence of another asymmetric species with one inward-oriented and two outward-oriented transport domains, but this structure could be refined only up to 4.8 Å resolution. Possibly, the collection and analysis of more particles would allow the determination of this structure at higher resolution, as well as the detection of Glt Tk in other states described by the binomial distribution of protomers over the apo and Na+-bound states, similar to what we will describe below for the substrate-unsaturated conditions.

Fully loaded state

At the concentration of 300 mM Na+ used in the experiments presented here, the apparent K d for l-aspartate is ~120 nM32, and therefore addition of 50 µM l-aspartate to 5.6 µM nanodiscs is expected to lead to substrate saturation. The cryo-EM structure of Glt Tk in the holo (Asp) state is symmetrical with all three transport domains in an intermediate-outward position (Figs. 1, 3a). This position is similar to the ones found in a protomer of Glt Ph (PDB 3V8G, chain C29; rmsd 0.965 Å, Supplementary Fig. 3b) and in the apo state described above, showing that the intermediate-outward orientation is visited by both the holo and apo transporters (Fig. 3b). In addition, the HP2 gates are closed in both cases, consistent with the ability to make elevator-type movements (Supplementary Table 1). Despite these similarities, there are also conspicuous differences. In the cryo-EM map of the holo (Asp) state, density for the amino acid substrate in the binding site is observed (Fig. 2b). Although the resolution is not high enough to unambiguously assign the density to aspartate, the positions of the binding residues are virtually the same as found in the crystal structure of Glt Tk with l-aspartate bound (PDB 5E9S)12. The conformation of residues involved in binding of the three Na+-ions also differs between the apo and holo (Asp) states, with the correct binding site geometries only adopted in the latter. Finally, the C-terminal half of TM7, the helices of HP2 and the N-terminal half of TM8 in the holo (Asp) state are displaced away from the center of the trimer by ~4 Å (Fig. 3b), which is another previously observed difference between the holo and apo states of Glt Tk and Glt Ph 11,12,33.

Fig. 3: Conformational differences of the Glt Tk protomers. a Comparison of the holo (Asp) Glt Tk cryo-EM (cornflower blue) and crystal (PDB 5E9S, light orange)12 structures (superposition using scaffold domains (yellow)) demonstrates differences between the fully outward and intermediate outward states. The panel below shows a slice through the transport domains in surface representation with aspartate molecules shown as sticks. The arrows indicate the movement of the transport domain from the fully-outward to intermediate-outward position. b Superposition on the scaffold domains of the cryo-EM structures of Glt Tk - holo (Asp) (cornflower blue) and Glt Tk -apo (cyan), which are both in intermediate-outward conformations. The lower panel shows the transport domains rotated 50° relative to the upper panel to highlight structural differences. c Superposition on the scaffold domains of cryo-EM structure of Glt Tk -TBOA (dark blue) and crystal structure Glt Tk -Asp (PDB 5E9S, light orange). The lower panel shows the transport domains rotated 40° relative to the upper panel with an arrow indicating opening of the HP2 gate. d Superposition of the cryo-EM structure of Glt Tk -Na+-only (light blue), the cryo-EM structure of ASCT2 (PDB 6RVX, light green)17, and a crystal structure of Glt Ph -Asp in the inward-oriented state (PDB 3KBC, pink)10. The lower panel highlights opening of the HP2 gate in the inward-oriented state. Superpositions on TM2 and TM5 of the scaffold domain. Full size image

Substrate unsaturated conditions

Using a sub-saturating aspartate concentration, in which we aimed to occupy ~1/3rd of the aspartate binding sites in the nanodisc preparation, we were able to solve two distinct structures, with different asymmetric arrangements of the transporter domains (Fig. 1). Roughly equal amounts of particles were used for the two reconstructions (Supplementary Fig. 1c). The individual protomers in the two structures are either in the inward-open state, identical to the state observed in the Na+-only condition, or in the outward-intermediate state, identical to the ones observed under aspartate-saturated conditions. The difference between the two structures is the number of protomers in each state, with either two inward and one intermediate-outward (2 in:1 out), or one inward and two intermediate-outward (1 in:2 out) oriented protomers. Our data indicate that we visualized a part of the multinomial distribution of the transport domains over all different possible states. The frequency of particles found with “2 in:1 out” or “1 in:2 out” arrangement (89% of all particles) indicates that the inward-open and holo outward-intermediate states had the highest probabilities of occurrence under the substrate-unsaturated conditions. It is possible that more states and a more complete multinomial distribution could be resolved if many more particles were collected.

TBOA-inhibited condition

In the four structures presented above, none of the protomers was in a fully outward orientation, which is remarkable, because this orientation is most frequently observed in crystal structures of both Glt Ph and Glt Tk . To test whether this state could be visited in our nanodisc preparations, we determined a structure in the presence of 120 µM DL-TBOA. This bulky competitive inhibitor of aspartate transport sterically prevents the closure of the HP2 gate, and traps the transporter in an open state, which in the crystal structures of Glt Ph is exclusively fully-outward (PDB 2NWW)9. Cryo-EM analysis of Glt Tk in this condition shows an asymmetric trimer with one transport domain positioned in an inward-oriented state and two domains in a fully-outward-state. The structure of the inward-oriented protomer is identical to the ones described above, and is similarly interpreted as representing a Na+-only state. The fully-outward protomers have wide open HP2 gates (distance between the tips of HP1 and HP2 13.8–14.5 Å), and show density for bound TBOA (Figs. 2d, 3c). The structures of these protomers are similar to those of crystal structures of Glt Ph in the presence of TBOA (rmsd 0.670 Å), and show that the fully-outward conformation is accessible in the nanodisc environment. This structure also reveals that the tip of HP1 of Glt Tk moves as much as 24 Å across the lipid bilayer in the transition between inward and outward states (Supplementary Table 2). The asymmetric Glt Tk structure with only two protomers occupied by the inhibitor suggests that the concentration of TBOA used was not saturating, which is consistent with the concentrations of Na+, DL-TBOA and protein used in the experiment, expected to lead to ~83% saturation.

Lipids and membrane scaffold protein

We selected the MSP2N2 belt protein for reconstitution, because it is able to form nanodiscs with large diameter (15 nm)37, however our structures show that their diameter is only ~10–11 nm, and the belt wraps around the protein more tightly (Supplementary Fig. 5). Nonetheless, we deduce from the structures that the transport domains had the freedom to move between inward and outward positions while embedded in the nanodiscs. All nanodisc samples were prepared in an identical way in the absence of substrates. The observation that redistributions of the transport domains to different conformations occurred upon addition of l-aspartate or DL-TBOA to the Glt Tk nanodiscs, shows that the belt protein did not prevent movement of the transport domains across the membrane.

Cryo-EM densities resembling phospholipids in a bilayer-like arrangement were found in the crevices between the protomers of the Glt Tk trimers, and indicate the position of the membrane (Fig. 4a). Since lipid-like densities were found only in these crevices, it was not possible to follow the shape of the lipid bilayer along the entire perimeter of the reconstituted protein. Therefore, we inspected the conformation of the MSP2N2 belt proteins, which were resolved in all structures obtained here. We used them as a guide to locate the position of the bilayer, also in places where lipid densities were not visible, and to estimate the extent of membrane deformations. We oriented the Glt Tk trimers with the z-axis along the pseudo three-fold axis of the scaffold domains, and then plotted the y-coordinates of the atoms from the modeled MSP2N2 protein as a function of the perimeter position (Fig. 4b). A straight horizontal conformation of the belt protein indicates a planar lipid bilayer, whereas buckling of the belt protein is indicative of membrane deformation. The straightest conformation of the MSP2N2 protein was found around the protomers with outward oriented transport domains, whereas the largest extent of buckling was found around the inward-oriented protomers. Apparently, buckling of the belt proteins did not lead to strongly disfavored conformations of Glt Tk in the nanodiscs, as in 6 out of the 15 protomers in the collective set of structures the transport domains are in inward positions. A recent molecular dynamics simulation on Glt Ph also showed perturbations of the lipid bilayer around inward-oriented transport domains of Glt Ph 38. The buckling of the belt proteins in our structures resembles these perturbations, indicating that the conformation of belt proteins provides at least an approximate position of the bilayer.