Transport mechanism of a bacterial homologue of glutamate transporters

Abstract

Glutamate transporters are integral membrane proteins that catalyze a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glial and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. The linchpin of the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm respectively. Here we describe a crystal structure of a double cysteine mutant of a bacterial homologue of glutamate transporters, Glt_Ph, which is trapped in the inward facing state by cysteine cross-linking. Together with the previously determined crystal structure of Glt_Ph in the outward facing state, the structure of the cross-linked mutant allows us to propose a molecular mechanism, by which Glt_Ph and, by analogy, mammalian glutamate transporters, mediate sodium-coupled substrate uptake.

Glutamate is the predominant excitatory neurotransmitter in the brain responsible for learning, memory formation and higher cognitive function. Specialized membrane proteins, glutamate transporters, also termed excitatory amino acid transporters (EAATs), mediate the transmitter uptake from the extracellular space into the cytoplasm of astrocytes and neurons against steep concentration gradients to allow for rounds of neurotransmission and to prevent glutamate-mediated excitotoxicity¹. EAATs are members of a ubiquitous SoLute Carrier 1 (SLC1) family of secondary solute transporters², which catalyze concentrative uptake of the acidic and neutral amino acids and dicarboxylic acids in a reaction coupled to symport of sodium and/or protons and, in the case of EAATs, antiport of potassium. The conceptual mechanisms of protein-mediated solute transport were developed over 40 years ago^3–5 and focus on the ability of transporters to undergo isomerization between two states: an outward facing state, in which the substrate-binding site is accessible from the external solution, and an inward facing state, in which it is reached from the cytoplasm. Although our understanding of the molecular mechanisms of transport have advanced significantly in the last decade^6,7, the structural transitions between the outward and inward facing states remain poorly characterized.

The sodium/aspartate symporter from Pyrococcus horikoshii (Glt_Ph), an archaeal homologue of the EAATs, was one of the first sodium-coupled transporters whose structure had been determined at a near atomic resolution^8,9. The structural analysis of Glt_Ph revealed that individual protomers assemble into a homotrimer, forming a deep solvent-filled bowl open to the extracellular solution and reaching approximately half way across the lipid bilayer (Fig. 1a). The first six transmembrane segments (TMs) of each Glt_Ph protomer mediate all inter-subunit contacts and form a distorted cylinder within which the highly conserved carboxyl terminus of the protein is folded into a compact core. The core consists of an intracellular re-entrant helical hairpin (HP) 1, TM7 with an unwound segment, an extracellular hairpin, HP2, and an amphipathic TM8. Despite the trimeric assembly, shared by all characterized glutamate transporter homologues^10–12, protomers function independently^13–17. Consistently, in Glt_Ph crystal structure an L-asp molecule and two sodium ions are bound within the core of each individual protomer. Positioned between the tips of HP1 and HP2 at the bottom of the extracellular bowl, the substrate is occluded from the aqueous milieu only by HP2. Strikingly, in a crystal structure of Glt_Ph in complex with a competitive blocker, L-threo-β-benzyloxyaspartate (L-TBOA)^9,18, HP2 assumes an open conformation, revealing its dynamic nature and suggesting that it may serve as an extracellular gate. Bound L-asp and L-TBOA are separated from the cytoplasm by over 15 Å of a compact protein core, suggesting that these structures correspond to the outward facing state of the transporter. However, the structure of the inward facing state and the manner in which the substrate and sodium ions are released into the intracellular solution, remain unknown.

Figure 1. Cross-linking of Glt_Ph-K55C/A364C.

a, Cartoon representation of two substrate-bound Glt_Ph protomers (PDB code 2NWX) viewed in the membrane plain. The third protomer and TM4 of the protomer on the right are removed for clarity. TMs 1 through 6 are coloured salmon and wheat; the carboxyl terminal cores are light blue; and the hairpins HP1 and HP2 are dark blue. Bound L-asp and Na⁺ are shown as sticks and purple spheres, respectively. Orange spheres correspond to the Cα atoms of residues 55 and 364. All molecular representations have been generated using Pymol⁴⁶. b, SDS PAGE analysis of Glt_Ph-K55C/A364C before and after incubation with 100 μM CuPhen for indicated periods of time. Detergent-solubilized purified Glt_Ph-K55C/A364C (left) and unpurified transporter in crude E. coli membranes (right) were visualized by Coomassie staining and western blotting, respectively. c, Cross-linking of Glt_Ph-K55C/A364C in the presence of 50 μM HgCl₂. Samples were analyzed as in b.

Cross-linking cysteines in TM2 and HP2

In a study predating the crystal structure determination of Glt_Ph, Vandenberg and colleagues reported that two cysteines placed in TM2 and HP2 of EAAT1 formed a spontaneous disulfide bond suggesting close proximity of these residues¹⁹. Strikingly, in the crystal structures of L-asp and L-TBOA-bound Glt_Ph the corresponding residues K55 in TM2 and A364 in HP2 are over 25 Å apart and cysteines at these positions are unlikely to become cross-linked. Moreover, the inter-subunit residue-to-residue distances are also 25–30 Å (Fig. 1a). Intrigued by these results, we reasoned that TM2 and HP2 become juxtaposed in an as yet structurally uncharacterized functional state of the transporter and set out to reproduce the double cysteine mutation in Glt_Ph for crystallographic studies. The K55C/A364C mutant was generated within a cysteine-less Glt_Ph, expressed in E. coli and purified in detergent solution. Oxidative cross-linking of Glt_Ph-K55C/A364C in the presence of copper 1,10-phenantroline (CuPhen) yielded distinct protein species with a higher electrophoretic mobility on SDS PAGE (Fig. 1b), which we identified as intramolecularly cross-linked Glt_Ph protomers (Supplementary Fig. 1a–c). Interestingly, CuPhen-catalyzed disulfide bond formation is greatly facilitated when the transporter is purified in the absence of sodium ions (Na⁺) and L-asp, suggesting that the state in which residues 55 and 364 are proximal, is more populated under these conditions (Fig. 1b and Supplementary Fig. 2a). Qualitatively similar results were also obtained using unpurified K55C/A364C mutant within E. coli crude membranes, demonstrating that Glt_Ph, like EAAT1, adopts the new conformation in the context of a lipid bilayer. Moreover, cysteines at structurally adjacent positions did not form a disulfide bond, suggesting that the distance shortening between positions 55 and 364 occurs in a highly specific manner (Ref. 19 and Supplementary Fig. 2b). Strikingly, incubation of Glt_Ph-K55C/A364C with divalent mercury (Hg²⁺), which serves as a bi-functional thiol-specific cross-linker, yields an essentially complete cross-linking both in the presence and in the absence of Na⁺ and L-asp in detergent and in crude membranes (Fig 1c). Furthermore, cross-linking is completed within seconds, suggesting that both substrate-free and bound Glt_Ph rapidly sample the crosslinking-competent state.

Crystal structure determination

Hg²⁺-cross-linked K55C/A364C mutant (Glt_Ph-55C/364C_Hg) in the presence of Na⁺ and L-asp yielded crystals diffracting to 3.5–3.9 Å (See Supplementary Table 1). A previous extensive study, conducted by Slotboom and colleagues, has suggested that the functionally relevant conformational transitions of a related bacterial glutamate transporter do not involve regions engaged in protein trimerization, because cross-linking of multiple interfacial residues had no effect on substrate transport¹⁷. Hence, we used a trimeric Glt_Ph model comprised of TM-s 2, 4 and 5, which are responsible for all inter-subunit contacts (Fig. 1a), to search for a molecular replacement solution. The initial phases yielded distinct peaks within the anomalous difference Fourier map corresponding to Hg atoms at positions adjacent to cysteines 55 in TM2. Rounds of manual building and refinement resulted in a protein model lacking 5 and 8 residues on the amino and carboxyl termini, respectively, and including 98 % of the side chains. To verify the model, we crystallized the selenomethionine-substituted Glt_Ph-55C/364C_Hg. The Se peaks within the anomalous difference Fourier maps are in perfect agreement with the location of 17 methionines in the protein model (Supplementary Fig. 3a). The distance between the Cα atoms of modelled cysteines 55 and 364 is 7.4 Å, and the Hg peak within the anomalous difference Fourier map is positioned between these residues with the sulphur to mercury distances estimated at 2 – 2.3 Å (Supplementary Fig. 3b), similar to those observed in small molecular weight compounds²⁰ and peptides^{21, 22}.

Structure of the inward facing state

The most striking structural feature of Glt_Ph-K55C/364C_Hg compared to the WT Glt_Ph, is a ca 18 Å movement of the substrate binding sites from near the extracellular solution at the bottom of the extracellular bowl, to near the cytoplasm (Figs. 2a, b). Structural superposition using trimerization TM-s 2, 4 and 5, along with the peripheral TM1 reveals that these segments remain largely unchanged, with the root mean square deviation (RMSD) for the atomic positions of 1.2 Å. The remainder of the protein, including TMs 3 and 6, HP1, TM7, HP2 and TM8, does not align and has undergone a substantial movement (Fig. 2a). Remarkably, when these regions alone are superposed, their structures are essentially identical, yielding RMSD of 0.6 Å. These results suggest that Glt_Ph can be partitioned into two structural domains, which we termed the trimerization and transport domains (Fig. 2c). The rigid body movements of the transport domains relative to the trimerization domain dominate the structural changes between the substrate-bound WT Glt_Ph and Glt_Ph-55C/364C_Hg (Fig. 2a,b and Supplementary Movie). The structural environment of the ion- and substrate-binding sites, which are positioned entirely within the transport domains, is preserved. The Fo-Fc difference Fourier map reveals excess electron density at these sites (Supplementary Fig. 4), suggesting that the transporter remains bound to L-asp and to two Na⁺ ions. Consistently, substrate-free Glt_Ph-55C/364C_Hg in solution binds L-asp with high affinity and in a sodium-dependent manner (Fig 2d). In the structure of Glt_Ph-55C/364C_Hg the substrate binding sites lie near the bottom of deep crevices formed within each protomer between HP1 and the cytoplasmic portion of TM7 of the transport domain and TMs 2 and 5 of the trimerization domain (Fig. 2b). Bound L-asp and Na1 are completely occluded from the solution by the tips of HP1 and HP2, while Na2 is partially exposed to the solvent (Fig. 2a, b). Because substrate and ion binding sites are near the intracellular solution, we propose that the conformation of Glt_Ph-55C/364C_Hg corresponds to an inward facing state of the transporter.

Figure 2. Glt_Ph-55C/364C_Hg in the inward facing substrate-bound state.

Cartoon representation of the single protomers (a) and surface representation of the trimers sliced through the binding sites (b). WT Glt_Ph and Glt_Ph-55C/364C_Hg are shown on the left and right, respectively. In (a), TMs 1, 2, 4 and 5 are coloured wheat with the remainder of the protomer light blue. Na⁺ are shown as purple spheres. c, Extracellular view of Glt_Ph-55C/364C_Hg with straight and curved lines delineating individual protomers and transport domains, respectively. The trimerization domains (wheat) and transport domains (blue) are connected by short cytoplasmic (solid arrows) and extracellular (open arrows) loops, highlighted in red. The long TM3–4 loops (simple arrows) cross over the transport domains. d, Isothermal titration calorimetry analysis of L-asp binding to Glt_Ph-55C/364C_Hg. Shown are the binding heats in 10 mM NaCl. The linear dependence (slope = −2.6 ± 0.7) of the log of the apparent dissociation constant, K_d, on the log of Na⁺ concentration is shown in the inset. Glt_Ph-55C/364C_Hg was exchanged into Na⁺/L-asp-free buffer, diluted to 15–20 μM in the reaction cell of the Microcal ITC200, supplemented with indicated concentrations of Na⁺, and titrated with L-asp at 25 °C. The binding enthalpy and the apparent number of binding sites were 23.6 ± 0.8 kcal/mol and 0.4 ± 0.03 (n=6), respectively.

Because the trimerization domain undergoes little conformational transitions, it is unlikely to change its position relative to the membrane plane. Hence, we estimate that a ~25 Å thick apolar region of the lipid bilayer, which in the WT Glt_Ph aligns approximately with the hydrophobic portion of TM1, is similarly positioned in Glt_Ph-55C/364C_Hg. Such bilayer placing suggests that several polar residues at the extracellular ends of the lipid-facing TMs 3 and 6 move into the hydrophobic region of the membrane. It is possible that the lack of the lipid bilayer in the crystals removes important constrains, resulting in a non-native positioning of the transport domain. However, cross-linking of residues 55C and 364C in Glt_Ph and corresponding residues in EAAT1 occurs efficiently in lipid membranes, suggesting that a sufficient range of motions of the transport domain is allowed. Movement of the transport domain toward the cytoplasm also significantly increases its exposure to the intracellular solvent. Strikingly, although the total exposed cytoplasmic surface area increases from ~1,300 in the WT to ~4,200 Ų in the Glt_Ph-55C/364C_Hg, the fraction of the apolar area changes only modestly from 57 to 63 %. Both values are well within the range reported for the solvent exposed area of soluble proteins²³.

Domain interaction interfaces

Both WT Glt_Ph and Glt_Ph-55C/364C_Hg bury approximately equal surface areas of ~2,500 Ų on the interface between the trimerization and transport domains. Furthermore, in both structures essentially the same residues of the trimerization domain are involved, forming a smooth relatively featureless interaction surface, which is over 80 % hydrophobic (Fig. 3a, b). Although TM1 and TM4 contribute to the contact area, TM2 and TM5, lying parallel to each other and crossing the membrane at an oblique angle, provide the bulk of the interactions, contributing 77 and 67 % to the interaction surface in the WT and Glt_Ph-K55C/A364C_Hg, respectively. In contrast, the transport domains in the two structures present interaction interfaces with little overlap, but similar in area (1250 Ų) and hydrophobicity (70%). They are dominated by HP1 and the cytoplasmic portion of TM7 in the WT Glt_Ph (Fig. 3a), and by HP2 and the extracellular portion of TM8 in Glt_Ph-55C/364C_Hg (Fig 3b), which provide 92 and 88 % of the total buried area, respectively. The analysis of the evolutionary conservation reveals that the interacting residues within the trimerization domain as well as within the two alternative interfaces of the transport domain are all highly conserved (Fig. 3c, d). The high conservation of HP2 surface residues and the reported transport inhibition of the mammalian EAATs upon chemical modification of cysteines in HP2^24–28 are consistent with this region being involved in specific protein contacts as observed in Glt_Ph-55C/364C_Hg. The disengagement of HP1 and the amino-terminal part of TM7 and their transition to the cytoplasmic surface of the transporter is also consistent with the previously reported intracellular solvent accessibility of these regions in related prokaryotic and mammalian transporters^29–32.

Figure 3. Domain interaction surfaces.

Trimerization domains of WT Glt_Ph (a) and Glt_Ph-55C/364C_Hg (b) are shown in surface representation and coloured wheat. Residues involved in domain contacts, identified by ProFace server⁴⁷, are coloured blue (TM1 and 2) and green (TM4 and 5). Interacting structural elements of the transport domain are shown in ribbon representation: HP1 (yellow) and TM7 (orange) in WT Glt_Ph, and HP2 (red) and TM8 (pink) in Glt_Ph-55C/364C_Hg. Surface representation of the trimerization (c) and transport (d) domains coloured according to evolutionary conservation. Dark blue and red correspond to the highly conserved and variable residues, respectively. The interacting surfaces are facing the viewer and the white arrows mark the highly conserved serine-rich signature motif in HP1. Conservation scores were calculated using Consurf server⁴⁸ and 212 SLC1 sequences with less than 60 % identity harvested from Pfam database⁴⁹ and aligned in ClustalW2⁵⁰.

It has been noted previously that the carboxyl terminal core of Glt_Ph contains structurally symmetrical elements⁸. Specifically, HP1 and the amino terminal half of TM7 can be superimposed on HP2 and the amino terminal portion of TM8 with RMSD of 2.5 Å. It is precisely these triplets of helices that constitute the two alternative interaction interfaces of the transport domain (Fig. 3a, b). Strikingly, also the amino terminal portion of the transporter, comprised of the first 6 TMs, exhibits a pseudo two-fold structural symmetry despite the lack of detectable sequence conservation. TMs 4 through 6 can be superimposed upon TMs 1 through 3 with RMSDs of ~6 and 4.3 Å for the WT and mutant, respectively (Fig. 4a, b). Hence, unlike other characterized secondary transporters^33–35, glutamate transporters contain not one, but two inverted structural repeats (Fig. 4d). Because of the amino-terminal symmetry, the interaction interface of the trimerization domain can be partitioned into structurally related extracellular and cytoplasmic portions (Fig. 4c), which contribute approximately equally to the total buried surface area.

Figure 4. Amino-terminal inverted structural repeat.

Cartoon representation of TMs 1–3 (blue) and TMs 4–6 (green) in the Glt_Ph-55C/364C_Hg viewed in the membrane plain (a), and their structural superposition (b). c, Symmetrical helices, TMs 1–2 and TMs 4–5, form the interaction surface within the transport domain, which is partitioned into intracellular and extracellular halves delineated by the dotted line. (d) Schematic representation of Glt_Ph trimerization (orange) and the transport (light blue) domains. Two inverted structural repeats are emphasized by blue and green, and yellow and red trapezoids. Structure of TM2–3 and TM5–6 loops in WT Glt_Ph (e) and Glt_Ph-55C/364C_Hg (f). TMs 2 through 6 are shown in cartoon representation with 4 omitted for clarity. The transporter core is shown in transparent surface representation. Bound L-asp and the highly conserved glycines are shown as spheres.

Hinge movements

Within the transport domain, structurally symmetrical TM3 and TM6 serve as two arms holding the transporter core and extending from the trimerization domain, the former from the cytoplasm and the latter from the extracellular solution (Fig. 4e, f). The loops, which connect these transmembrane segments to the trimerization domain, enable the inward movement. The long flexible loop between TMs 3 and 4 is poorly structured in the L-asp-bound WT Glt_Ph crystals, and in Glt_Ph-55C/364C_Hg its structure is largely determined by crystal contacts. In contrast, the structurally symmetrical loops between TMs 2 and 3 and between TMs 5 and 6 are short and undergo well-defined transitions to accommodate the movements of the transport domain (Fig. 4e, f and Supplementary Movie). In Glt_Ph-55C/364C_Hg, unwinding of the TM3 α-helix by approximately one turn extends TM2–3 loop by 4 residues and allows for the descent of TM3 toward the cytoplasm. In contrast, the TM5–6 loop is shortened by 2 residues as a result of simultaneous unwinding of TM5 by a half of a turn and extension of TM6 by a turn of a helix. Both loops contain conserved glycines, G69, G79 and G82 in TM2–3 loop, and G221 in TM5–6 loop, which may facilitate the folding/unfolding of the helices and serve as hinges. Indeed, the transport domain movement can be decomposed into a 16 Å descent toward the cytoplasm followed by a 37 ° rotation around an axis passing simultaneously through the transport domain centre of mass and TM2–3 and TM5–6 loops. Consistent with the importance of the structural rearrangements in the loop regions, chemical modifications of cysteines in TM5–6 loop of EAAT3 inhibited uptake³⁶.

Transport mechanism

In the structure of the WT Glt_Ph, L-asp is bound ~5 Å beneath the surface of the extracellular bowl occluded from the solution by HP2. In contrast, in the structure of Glt_Ph-55C/364C_Hg, L-asp is bound ~5 Å beneath the intracellular surface occluded from the solution by HP1. Hence, we propose that L-asp-bound Glt_Ph structure corresponds to the state right after binding of the substrate from the extracellular side, outward-facing occluded, and Glt_Ph-55C/364C_Hg structure - to the state right before the release of the substrate into the cytoplasm, inward-facing occluded (Fig 5). The isomerization between these states involves a combination of a cross-membrane movement and rotation of the transport domain, comprised of the substrate-binding transporter core and peripheral TMs 3 and 6. During this movement, the lipid-facing hydrophobic TMs 3 and 6 traverse the bilayer directly, moving toward the cytoplasm by ~12 Å. In contrast, passage of the relatively polar HP1 and HP2, with their tips crossing as much as 20 Å of the lipid bilayer, is facilitated by the intra-protein track provided by the trimerization domain. The transport domains are expected to move stochastically and independently within each protomer, so that the crystal structure represents a statistically rare case, when all three transport domains are in the inward orientation. The rigid trimerization domain is anchored in the membrane and provides a counterbalance to the movements of the bulky transport domains, suggesting an explanation for the obligatory oligomeric assembly of SLC1 family of transporters.

Figure 5. Schematic transport mechanism.

Shown is a single transporter protomer. Substrate and sodium binding to the outward and inward facing states is coupled to the closure of the extracellular and intracellular gates, HP2 (red) and HP1 (yellow), respectively. Isomerization between the outward and inward facing occluded states occurs upon movement of the transport domain (blue), relative to the trimerization domain (grey). The inward facing open state has not been structurally characterized and is hypothetical.

Substrate binding/unbinding on either side of the membrane is associated with additional conformational changes, or gate openings. The crystallographic data and molecular dynamic simulations^{9, 37, 38} suggest that substrate and ion dissociation on the extracellular side is associated with the opening of HP2, defining it as an extracellular gate. We hypothesize that in a functionally symmetrical manner, movements of HP1 allow for dissociation of the substrate and ions from the inward facing state, defining it as a bona fide intracellular gate. The alternate opening of the extracellular and intracellular gates is strictly maintained: in the outward facing state, when HP2 opens to expose the substrate and ion binding sites to the extracellular solution, HP1 is secured in the closed state, packed against TMs 2 and 5. Conversely, in the inward facing state HP2, the extracellular gate, is locked closed upon displacing HP1.

As has been suggested previously³⁹, coupling of the substrate transport to the energy of the ionic gradients is established via synergistic binding of substrate and sodium ions on the extracellular and intracellular sides of the membrane coupled to the closure of the corresponding gates. The steep sodium dependence of the substrate binding, observed both in WT Glt_Ph⁹ and in Glt_Ph-55C/364C_Hg (Fig. 2d), is responsible for the differential affinity for the substrate on the extracellular side of the membrane, where sodium concentration is high, and the intracellular side, where it is low. We further propose that the isomerization between the outward and inward facing states is sodium independent and may simply be driven by the thermal energy alone. The observation that Hg²⁺-mediated cross-linking of K55C/A364C mutant is completed within seconds both in membranes and in detergent suggests that this transition is rapid and does not limit the rate of transport in Glt_Ph, which has a turnover time of ~3 minutes at ambient temperatures⁴⁰. Finally, we hypothesize, that a similar isomerization reaction may also occur in the apo-transporter to complete the transport cycle. The structural re-arrangements within the transport domain, which would allow for the closure of the extracellular and the intracellular gates in the absence of bound substrate and ions, remain to be elucidated.

Methods

Cysteine cross-linking

Glt_Ph-K55C/C321A/A364C was expressed as His₈ fusion and purified as described previously⁸. Transporter samples were exchanged by size exclusion chromatography (SEC) into buffer, containing (in mM) 10 HEPES/NaOH or KOH, pH 7.4, 1 n-dodecyl-β-D-maltopyranoside and either 100 NaCl and 0.1 L-asp or 100 KCl. Cross-linking was initiated by addition of 1:2 molar ratio of Cu²⁺ and 1,10 phenantroline or HgCl₂. Reactions were quenched with 100 mM N-ethyl maleimide prior to SDS PAGE analysis. Crude E. coli membranes were isolated by centrifugation, washed either in a Na⁺/L-asp-containing or free buffer and cross-linked as in detergent. Protein bands were visualized by western blotting using antibodies against histidine tag.

Crystallography

K55C/C321A/A364C mutation was introduced within a heptahistidine mutant of Glt_Ph, used in earlier crystallographic studies^8,9, to which we refer as “wild type” for brevity. Purified protein was cross-linked in the presence of 10 fold molar excess of HgCl₂, dialyzed against buffer containing 10 mM HEPES/NaOH, 7 mM n-decyl-β-D-maltopyranoside, 100 mM NaCl and 100 μM L-asp, diluted to the final concentration of 2–4 mg/ml and supplemented with 0.5 mM E. coli total polar lipid extract and 100 mM NaBr. Protein solution was mixed at 1:1 (v:v) ratio with the reservoir solution, containing 100 mM MES, pH 5.0, 18–20 % PEG 350 MME and 200 mM CaCl₂, and crystallized at 4 °C by hanging drop vapour diffusion. Crystals were cryoprotected by allowing the drop to dry until its volume was reduced by 50 %. Selenomethionine-substituted protein was expressed as described previously⁸ and crystallized as above. Diffraction data were indexed, integrated and scaled using HKL-2000 package⁴¹. Further analyses were performed using CCP4 programs⁴². Initial phases were obtained using Phaser⁴³, and the protein model built manually in Coot⁴⁴ and refined using REFMAC⁴² with TLS⁴⁵ and three fold NCS restrains.