Tuning Activation of the AMPA-Sensitive Glur2 Ion Channel by Genetic Adjustment of Agonist-Induced Conformational Changes

Tuning activation of the AMPA-sensitive GluR2 ion channel by genetic adjustment of agonist-induced conformational changes

Neali Armstrong*, Mark Mayer†, and Eric Gouaux*‡§

*Department of Biochemistry and Molecular Biophysics and ‡Howard Hughes Medical Institute, Columbia University, New York, NY 10032; and †Laboratory of Cellular and Molecular Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved March 17, 2003 (received for review December 5, 2002)

The (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptor discriminates between agonists in terms of binding and channel gating; AMPA is a high-affinity full agonist, whereas kainate is a low-affinity partial agonist. Although there is extensive literature on the functional characterization of partial agonist activity in ion channels, structure-based mechanisms are scarce. Here we investigate the role of Leu-650, a binding cleft residue conserved among AMPA receptors, in maintaining agonist specificity and regulating agonist binding and channel gating by using physiological, x-ray crystallographic, and biochemical tech- niques. Changing Leu-650 to Thr yields a receptor that responds more potently and efficaciously to kainate and less potently and efficaciously to AMPA relative to the WT receptor. Crystal structures of the Leu-650 to Thr mutant reveal an increase in domain closure in the kainate-bound state and a partially closed and a fully Fig. 1. Mechanisms to describe the conformational behavior of ligand-gated closed conformation in the AMPA-bound form. Our results indicate ion channels. (A) The two-state model where the receptor is in equilibrium that agonists can induce a range of conformations in the GluR2 between two conformations, closed and open. Agonist binding stabilizes the ligand-binding core and that domain closure is directly correlated receptor in the open state. Full agonists stabilize the open state more effec- to channel activation. The partially closed, AMPA-bound confor- tively than partial agonists, and both types of agonists stabilize the same conformational states. (B) Multistate model where the agonist-binding region mation of the L650T mutant likely captures the structure of an of the receptor adopts a range of agonist-dependent conformations. Partial agonist-bound, inactive state of the receptor. Together with pre- agonists promote a submaximal conformational change and therefore are not viously solved structures, we have determined a mechanism of as effective in shifting the closed to open equilibrium of the ion channel to the agonist binding and subsequent conformational rearrangements. open state.

igand-gated ion channels are allosteric proteins composed of Lagonist binding and ion channel domains (1). Agonists do agonist-binding domain and activation of the ion channel by work on the ion channel by coupling the energy derived from ‘‘tuning’’ ion channel gating via a specific combination of a agonist binding to the opening or gating of the ion channel. By partial agonist and a site-directed mutant in the agonist binding site. definition, full agonists produce maximal activation of the ion AMPA receptors (GluR1–4) are a subtype of the ionotropic channel, whereas partial agonists result in submaximal activa- glutamate receptor family of ligand-gated ion channels (7, 8) and tion, even when applied at saturating concentrations. There are have a high affinity for the full agonist AMPA and a low affinity two distinct models to describe the behavior of allosteric proteins for the partial agonist kainate (9–11). AMPA receptors also such as ligand-gated ion channels: a two-state or concerted bind and activate in response to the nonselective, full agonists model (2) and an induced-fit or multistate model (3, 4) (Fig. 1). L-glutamate and quisqualate (9–12). Crystallographic studies In the two-state model, the ligand-gated ion channel exists in reveal that full and partial agonists bind to the cleft of the two conformations, an inactive or ‘‘apo’’ conformation and an ‘‘clamshell-shaped’’ GluR2 S1S2J ligand-binding core (12–15): agonist-bound, activated conformation. Full and partial agonists The full agonists AMPA, glutamate, and quisqualate bring the can bind to both states, with full agonists more effectively domains of the ligand-binding core Ϸ21° closer together, relative stabilizing the activated state in comparison with partial agonists to the apo state, whereas the partial agonist kainate induces only (5). Thus, full agonists produce greater activation of the ion 12° of domain closure (14). Kainate induces only partial domain channel than partial agonists. Cyclic-nucleotide gated ion chan- closure because its isopropenyl group acts like a ‘‘foot in the nels from bovine rod photoreceptors respond maximally to door,’’ colliding with Tyr-450 and Leu-650. On the basis of these cGMP but only weakly to cAMP and are paradigms of the structural studies, we suggest that the ligand-binding core can two-state model (5, 6). adopt multiple, agonist-dependent conformations and that dif- According to the multistate hypothesis, the ligand-gated ion ferences in agonist efficacy at AMPA receptors can arise from channel can adopt a range of conformations that depends on the different conformations of the ligand-binding core. particular agonist (Fig. 1B). Full agonists stabilize a conformation that maximally activates the ion channel and partial agonists stabilize different conformations that are less efficacious in This paper was submitted directly (Track II) to the PNAS ofﬁce. channel activation; i.e., for partial agonists less binding energy is Abbreviations: AMPA, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid; AS, available for doing work necessary to open the ion channel gate. ammonium sulfate; Imax, maximal current measured at saturating agonist concentration. Using the GluR2 (S)-2-amino-3-(3-hydroxy-5-methyl-4- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, isoxazole) propionic acid (AMPA)-sensitive ion channel, we www.rcsb.org (PDB ID codes 1P1N, 1P1O, 1P1Q, 1P1U, and 1P1W). have studied the relationships between the conformation of the §To whom correspondence should be addressed. E-mail: [email protected].

5736–5741 ͉ PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1037393100 Downloaded by guest on September 29, 2021 To test this hypothesis, we reasoned that if the steric clash a reservoir solution containing 24–28% polyethylene glycol between the isopropenyl group of kainate and neighboring (PEG) 4000 and 0.2–0.35 M ammonium sulfate (AS). The two residues in the ligand-binding core were reduced, then kainate crystal forms for the S1S2J L650T͞AMPA and L650T͞ binding should induce greater domain closure and therefore be quisqualate complexes were obtained with 12–16% PEG 8000, able to do more work on the ion channel; i.e., kainate should 0.1–0.3 M zinc acetate, and 0.1 M cacodylate, pH 6.5 (Zn form), become an agonist with greater efficacy. Because mutation of or with 14–18% PEG 4000 and 0.2–0.4 M AS (AS form). S1S2J the conserved Tyr-450 residue to smaller residues resulted in L483Y͞L650T was cocrystallized with 20 mM AMPA by using nonfunctional ligand-binding core protein, we focused our stud- 14–18% PEG 4000 and 0.2–0.4 M AS as precipitant. Crystals ies on residue 650. In fact, mutation of the equivalent residue in were cryoprotected with mother liquors supplemented with GluR1, Leu-646 to Thr (L646T), produces a decrease in the 14–18% glycerol before flash cooling in liquid nitrogen. kainate EC50 and an increase in the extent of kainate current potentiation by cyclothiazide, suggesting that kainate is both a Crystallography. All data sets were collected at the National more potent and strongly desensitizing agonist when acting on the L646T mutant, in comparison with the WT receptor (16). In Synchrotron Light Source beamline X4A (Upton, NY) by using a Quantum 4 charge-coupled device detector except for the addition, leucine is conserved at position 650 in AMPA recep- ͞ ϫ tors, whereas in kainate receptors, which respond maximally to L650T AMPA (Zn form) data set, which was collected at 25 kainate but weakly to AMPA, the residue is either a valine or an on a Brandeis B4 charge-coupled device detector. Data sets were isoleucine. Here we report complementary functional and struc- indexed and merged by using the HKL suite (21). The S1S2J ͞ tural studies of the Leu-650 to Thr (L650T) mutant of the GluR2 L650T kainate, AMPA (AS form), quisqualate (AS form), and receptor, illuminating relationships between agonist binding, the S1S2J L483Y͞L650T͞AMPA structures were solved by domain closure, and ion channel activation. molecular replacement using AMORE (22) with the S1S2J kainate protomer, S1S2J AMPA dimer, S1S2J quisqualate monomer, Materials and Methods and S1S2J AMPA dimer structures as the search probes, re- Molecular Biology. The S1S2J constructs (14) were derived from spectively (12, 14). The S1S2J L650T͞quisqualate(Zn) and the GluR2 (flop) gene (17), whereas the unedited GluR2 or L650T͞AMPA(Zn) crystal forms were refined beginning from GluRB (flip) gene (10), in the pGEM-HE expression vector (32), the WT structures by using X-PLOR (23) and CNS (24). The was used in the physiology. The L650T mutation was incorpo- protocols included rigid-body refinement, simulated annealing, rated into the GluR2 S1S2J, GluR2 S1S2J L483Y, and full- Powell minimization, individual B-value refinement, and bulk- length constructs (14, 18) by using the QuikChange protocol solvent modeling. After every round of refinement, the model (Stratagene) and the primers 5Ј-ATGGAACAACCGACTCT- Ј Ј was manually compared with the electron density by using omit GGATCCACTAAAG-3 and 5 -GTGGATCCAGAGTCGGT- maps, and the model was manually fit to the electron density. TGTTCCATAAGCA-3Ј. The correct clones were confirmed by Refinement continued until the crystallographic R factors con- sequencing both strands of the DNA. verged for the S1S2J L650T͞kainate, quisqualate (AS form), and AMPA (AS form) structures. Refinements of the S1S2J Physiology. The ovaries of tricaine (3 g͞liter)-anesthetized Xe- L650T͞AMPA (Zn form), quisqualate (Zn form), and nopus laevis were surgically removed and placed in 30 ml of ͞ ͞ Barth’s solution, cut into small pieces, and incubated for 60 min S1S2J L483Y L650T AMPA structures were carried out until it at 25°C with 1.5 mg͞ml collagenase. Defolliculated oocytes were was clear that the refined structures were essentially identical to rinsed and stored in 88 mM NaCl, 2.5 mM NaHCO3, 1.1 mM the starting model. KCl, 0.4 mM CaCl2, 0.3 mM Ca(NO3)2, 0.8 mM MgCl2, 2.5 mM sodium pyruvate, 10 mM Hepes, pH 7.3, and 5 ␮g͞ml gentamicin Agonist Affinity Measurements. Measurements of agonist Kd values at 18°C. Stage V and VI oocytes were injected with 0.5–2.0 ng of were carried out by fluorescence spectroscopy using previously GluR2-L483Y, GluR2-L483Y͞L650T, GluR2-L650T, or WT established methods (25, 26). The protein was dialyzed exten- GluR2 in vitro-transcribed cRNA, and recordings were done 3–5 sively against 10 mM sodium phosphate (pH 7.4) to reduce the days later. concentration of glutamate. Three milliliters of freshly filtered Recordings were performed in the two-electrode voltage- protein was placed in a quartz cuvette maintained at 4°Cbya clamp configuration by using agarose-tipped microelectrodes circulating water bath. The protein concentration used varied by (0.4–1.0 M⍀) filled with 3 M KCl. The holding potential was Ϫ60 agonist (0.05–1 ␮M) so as to keep the protein concentration at mV. The bath solution contained 100 mM NaCl, 1 mM KCl, 0.7 least 2-fold below the Kd. The excitation wavelength was 280 nm mM BaCl2, 0.8 mM MgCl2, and 5 mM Hepes, pH 7.3. Rundown and emission was detected at 330 nm with bandpasses of 4 nm was corrected by measuring the response to a saturating con- and 8 nm, respectively. Small aliquots of agonist (1–10 ␮l) were centration of glutamate in the beginning, middle, and end of the added to the desired final concentration and the total volume of agonist applications. Cyclothiazide was added from a 0.1 M added ligand was Ͻ1%. The small dilution of starting solution stock, in DMSO, to both bath and agonist solutions for a final was ignored. After each addition of agonist, the sample was concentration of 100 ␮M. For the maximal current measured at thoroughly mixed by pipetting and the fluorescence at 330 nm saturating agonist concentration (Imax) experiments, all agonists were applied to the same oocyte, and the responses elicited by was measured. The fluorescence change at each agonist con- glutamate were normalized to 1.0. In experiments that required centration was measured five times and recorded as the average. concentrations of agonist Ͼ1 mM, a uniform osmolarity was The fluorescence at each point was calculated as maintained by addition of sodium chloride to solutions with Ϫ Fo Fn lower concentrations of agonist. The data were acquired and ⌬F ϭ , processed by using the program SYNAPSE. Dose–response curves Fo were plotted and fit to the Hill equation in KALEIDAGRAPH. where Fo is the fluorescence from protein alone and Fn is the Crystallization. The GluR2 S1S2J L650T and S1S2J L483Y͞ fluorescence after the nth addition of ligand. The ⌬Fs were fit L650T proteins were expressed and purified as described (19, to the Hill equation, with the Hill coefficient set equal to 1.0, by BIOPHYSICS 20). All crystals were grown by hanging drop vapor diffusion at using KALEIDAGRAPH. Measurements were performed in dupli- 4°C. S1S2J L650T was cocrystallized with 20 mM kainate over cate or triplicate.

Armstrong et al. PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 5737 Downloaded by guest on September 29, 2021 reduction in affinity of the GluR2 ligand-binding core for glutamate, quisqualate, and AMPA upon substitution of Leu- 650 for Thr results, at least in part, from loss of nonpolar contacts made by the leucine side chain that span the cleft between domain 1 and domain 2 (13, 14). Substitution of the leucine by a smaller, polar side chain precludes formation of similar interactions, destabilizing the agonist-bound, closed-cleft conformation of the ligand-binding core. As described below, the L650T mutation increases kainate affinity because it allows for further domain closure, relative to the WT ligand-binding core. To test whether the substitution of leucine by threonine at position 650 altered the ability of glutamate, quisqualate, AMPA, or kainate to activate the ion channel, we measured the maximal current elicited by saturating concentrations of agonist (Imax). As shown in Fig. 2, L483Y receptors had similar maximal responses to glutamate, quisqualate, and AMPA, whereas the kainate-induced currents were 2.0% of those elicited by glutamate. L483Y͞L650T receptors also had similar I values for Fig. 2. Dose–response curves and Imax traces for the L483Y and L483Y͞L650T max variants of the full-length GluR2 receptor recorded by using the two- glutamate and quisqualate. By contrast, the currents evoked by electrode, voltage–clamp technique. (A) Normalized dose–response curves kainate in the L483Y͞L650T receptor were 24% as large as those for glutamate (Glu) (}), AMPA (■), quisqualate (Quis) (Œ), and kainate (KA) (F) elicited by glutamate, suggesting that kainate is an Ϸ10-fold measured from oocytes expressing GluR2 L483Y receptors. (B)EC50 data for more efficacious agonist upon reduction of the size of the side the GluR2 L483Y͞L650T mutant, in combination with glutamate (}), AMPA chain at 650 from a leucine to a threonine. ■ Œ F ( ), quisqualate ( ), or kainate ( ), scaled for efﬁcacy relative to glutamate. Surprisingly, the L650T mutation changes AMPA to a partial (C) Maximal currents (Imax) elicited by L483Y receptors and saturating concen- ͞ trations of agonist. The duration of agonist application is indicated by hori- agonist, and, in the context of L483Y L650T, saturating con- zontal, thick black lines, and the identity and concentration of the agonist is centrations of AMPA only activate the receptor to 38% of the indicated above each line. (D) Maximum currents mediated by L483Y͞L650T maximal response produced by glutamate and quisqualate. To ͞ receptors. The Imax measurements were made on ﬁve individual oocytes. All ensure that the L483Y and L483Y L650T Imax data were not values are summarized in Table 1. distorted by the L483Y mutation, we repeated the experiments with the WT GluR2 receptor and the L650T point mutant, using cyclothiazide to block desensitization (Table 1; refs. 11, 28, and Results and Discussion 29), and we obtained qualitatively similar results. L650T Inverts the Relative Potency of AMPA and Kainate Responses. Nevertheless, we did find a substantial difference between the To measure the extent of receptor activation, we used the relative Imax measurements of kainate-elicited currents in the nondesensitizing variant of GluR2, the Leu-483 to Tyr (L483Y) context of L483Y versus the WT receptor plus cyclothiazide. As mutant (18, 27) as the WT species and introduced the L650T shown in Table 1, kainate results in currents that are 17% of the mutation in the L483Y background (L483Y͞L650T) for two- glutamate currents for the WT receptor͞cyclothiazide combi- electrode voltage–clamp experiments. Dose–response analysis nation, whereas kainate acting on the L483Y variant yields of the L483Y and L483Y͞L650T receptors in the presence of currents that are 2% of those evoked by glutamate. Therefore, kainate, glutamate, AMPA, or quisqualate (Fig. 2) shows that we suggest that cyclothiazide may more effectively block desen- the L650T mutation increases the EC50 values, decreasing the sitization in comparison with the L483Y mutant. Indeed, pre- potencies of glutamate, quisqualate, and AMPA 8.5-, 66-, and vious experiments carried out on native heteromeric AMPA 70-fold, respectively, relative to L483Y (Table 1). By contrast, receptors have shown that kainate currents are Ϸ37% of the ␮ the EC50 for kainate decreased 2.7-fold from 173 to 65 M, magnitude of glutamate currents in the presence of cyclothiazide indicating an increase in potency. The shifts in EC50 values were (11), and fast-perfusion measurements of kainate and glutamate mirrored by changes in Kd values for binding of the same ligands activation of GluR3-L507Y (18, 27) showed that the ratio of peak to the isolated L650T ligand-binding core: The Kd values for kainate to peak glutamate currents ranged from Ϸ0.13 to Ϸ0.25. glutamate, quisqualate, and AMPA increase 13-, Ϸ170-, and These data provide additional support for the conclusion that in Ϸ 500-fold, respectively, whereas the Kd for kainate decreases the oocyte experiments reported here the agonist responses in Ϸ12-fold (see Table 1 and Fig. 7, which is published as support- the context of the L483Y mutant may be submaximal because of ing information on the PNAS web site, www.pnas.org). The a modest extent of receptor desensitization.

Table 1. Agonist potency, efﬁcacy, and afﬁnity WT L650T

L483Y CTZ Afﬁnity, L483Y CTZ Afﬁnity,

Agonist EC50, ␮M Hill efficacy efficacy ␮MEC50, ␮M Hill efficacy efficacy ␮M

Glu 20.8 Ϯ 0.64 1.82 1.00 1.00 0.821* 177 Ϯ 9.2 1.77 1.00 1.00 6.61 Ϯ 0.58 Quisqualate 1.18 Ϯ 0.10 1.85 0.98 1.01 0.010† 78.2 Ϯ 1.0 1.61 1.04 1.01 1.74 Ϯ 0.05 AMPA 5.88 Ϯ 0.12 1.37 0.99 1.16 0.025* 412 Ϯ 26.4 1.37 0.38 0.31 12.8 Ϯ 0.56 Kainate 173 Ϯ 9.02 1.21 0.02 0.17 1.94 64.7 Ϯ 4.1 1.21 0.24 0.45 0.16 Ϯ 0.02

EC50 values and Hill coefﬁcients were calculated from dose–response curves measured from GluR2-L483Y or GluR2-L483Y͞L650T. To calculate relative agonist efﬁcacy, the maximal current response was scaled to the value for glutamate, which was normalized to 1.0. *For the WT GluR2 S1S2J ligand-binding core where the value for AMPA is the Kd and the values for glutamate and kainate are IC50 measurements in competition with 3H AMPA (14). † 3 IC50 value for the WT GluR2 S1S2J ligand-binding core in competition with H AMPA (30).

5738 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1037393100 Armstrong et al. Downloaded by guest on September 29, 2021 Table 2. S1S2J L650T reﬁnement statistics rms deviations

B values No. protein † Complex dmin Rwork* Rfree atoms No. water Mean B Bonds, Å Angle, ° Bond Angle Kainate 1.6 23.5 25.2 1,918 123 18.3 0.0062 1.41 1.18 1.83 AMPA(AS) 2.0 23.7 27.2 3,868 177 15.9 0.0055 1.24 1.39 1.98 AMPA(Zn) 2.0 21.7 27.0 5,891 421 16.7 0.0065 1.31 1.20 1.95 Quisqualate(AS) 1.6 20.4 23.5 1,953 220 11.2 0.0052 1.19 1.10 1.80 L483Y-AMPA 1.8 23.4 26.4 3,875 235 17.8 0.0055 1.22 1.38 1.84

*Rwork ϭ (͚ ʈFo͉ Ϫ ͉Fcʈ)͚͞ ͉Fo͉, where Fo and Fc denote observed and calculated structure factors, respectively. † Ten percent of the reﬂections in each data set was set aside for the calculation of the Rfree value.

L650T Increases Domain Closure Produced by Kainate. To determine unit related by a noncrystallographic twofold axis, one of which the effect of the L650T mutation on the structure of the ligand- adopts the partially (Ϸ11°) closed conformation (molecule B) binding core, we determined crystal structures of the kainate, whereas the other is fully closed (molecule A), similar to the WT AMPA, and quisqualate complexes by x-ray crystallography and S1S2J͞AMPA complex. Illustrated in Fig. 4 are superpositions compared them with the corresponding structures of the WT ligand-binding core. The structures of all complexes were determined to a resolution of 2.0 Å or higher (see Table 3 and Figs. 8 and 9, which are published as supporting information on the PNAS web site) and were subject to thorough refinement, as summarized in Table 2. As predicted by the physiology data, the extent of domain closure in the L650T S1S2J͞kainate complex is greater than the degree of domain closure in the WT S1S2J͞ kainate complex. Shown in Fig. 3 is a superposition of the L650T and WT kainate cocrystal structures. We find that the L650T͞ kainate structure is 15° more closed than the WT Apo(A) structure and 5° more open than the WT AMPA(A) structure (14). By contrast, the WT S1S2J͞kainate structure is 12° more closed than Apo(A) and 8° more open than AMPA(A) (14). The L650T mutation allows for greater domain closure in the kainate complex because the smaller threonine side chain min- imizes the steric clash between the isopropenyl group of kainate and the larger leucine side chain present in the WT receptor. Specifically, the ␣-carbon of Thr-650 moves Ϸ0.8 Å farther into the binding cleft relative to the ␣-carbon of Leu-650. The relocation of Thr-650 is coupled to a rigid body movement of most of domain 2, resulting in a 3° increase in domain closure in the L650T͞kainate complex, relative to the WT S1S2J͞kainate structure. Because the ligand-binding cores of AMPA receptors are arranged as back-to-back dimers, domain closure is coupled to an increase in the distance between the ‘‘linker’’ regions, proximal to the ion channel gate (18). Therefore, the increase in domain closure in the L650T͞kainate complex translates into an increase in linker separation of Ϸ1.0 Å, relative to the WT kainate complex (Fig. 3C). We suggest that in the intact L483Y͞ L650T receptor, kainate binding results in greater domain closure, together with greater linker separation, and this in turn is coupled to an increase in efficacy of channel gating. Deter- mining whether the increase in kainate induced currents in the Fig. 3. Comparison of the WT and S1S2J L650T͞kainate conformations. (A) ͞ L483Y͞L650T receptor, relative to glutamate and quisqualate, is Superimposition of WT and L650T kainate structures. The WT backbone is drawn in gray and the mutant structure is shown in magenta. Kainate is drawn caused by an increase in open probability or single-channel in black. Selected binding cleft side chains are drawn in ball-and-stick repre- conductance, or both, will require in-depth single-channel sentation and colored as the backbone. The large spheres at the bottom of the analysis. structure depict the location of the ﬁrst residue (Gly) in the two residue Gly-Thr linker. (B) Close-up view of superimposed WT and mutant kainate (KA) AMPA Is a Partial Agonist in the Context of L650T. To address this binding sites. The green sphere is a water in the mutant structure and the unexpected result, we analyzed crystal structures of the L650T purple sphere is a water in the WT structure. In the mutant structure, the mutant in complexes with AMPA or quisqualate. Strikingly, the hydroxyl of Thr-650 makes a water-mediated hydrogen bond with kainate ͞ crystallographic analysis of multiple crystal forms reveals that that is absent in the WT structure. In the L650T kainate structure, two key residues situated on the clamshell cleft move 0.6 Å closer together relative to the L650T͞AMPA complex adopts multiple conformations that Ϸ the WT kainate structure, placing the hydroxyl of Thr-686 in ideal hydrogen range from partially closed with 11° of domain closure relative bonding distance (2.6 Å) to a carboxylate oxygen of Glu-402. (C) Superimposed to the WT Apo subunit to fully closed conformations with 21–22° S1S2J͞kainate and L650T͞kainate crystallographic dimers. WT protomers are BIOPHYSICS of domain closure. In the cocrystals of L650T͞AMPA grown in drawn in gray and mutant protomers are shown in magenta (A) and orange the presence of AS, there are two molecules in the asymmetric (A*-symmetry related protomer). Thr-650 is drawn in space-ﬁlling mode.

Armstrong et al. PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 5739 Downloaded by guest on September 29, 2021 Fig. 4. Comparison of WT and S1S2J L650T͞AMPA(AS) conformations. (A) Superposition of WT S1S2J͞AMPA (gray) with S1S2J L650T͞AMPA (AS form) protomer A (blue). (B) Superposition of WT S1S2J͞AMPA (gray) with S1S2J L650T͞AMPA(AS) protomer B (green). The black arrows in A and B indicate the axis of rotation relating the conformational difference between the WT and L650T structures. (C) Superimposed WT and mutant AMPA dimers.

of the WT S1S2J͞AMPA and L650T S1S2J͞AMPA structures. full domain closure and are similar to the previously determined To determine whether other L650T͞AMPA conformations WT quisqualate structure (data not shown). Therefore, with could be observed in different crystal forms, we solved the respect to domain closure, the L650T͞quisqualate structure is structure of a second crystal form that contains three molecules similar to the WT AMPA and glutamate structures. in the asymmetric unit (Zn form), and we crystallized and solved Our crystallographic analyses suggest that AMPA is a partial the structure of the L483Y͞L650T S1S2J͞AMPA double mu- agonist at the L650T receptor, relative to glutamate and quis- tant, which has two molecules in the asymmetric unit. For both qualate, because the L650T mutation has dramatically destabi- of the crystal forms, the extent of domain closure in the lized the closed-cleft, AMPA-bound conformation. This, in turn, ligand-binding core was 21–22°, similar to the WT AMPA allows for the L650T͞AMPA receptor to populate conforma- complex. tions that have different, submaximal degrees of domain closure. To directly compare the crystallographic studies of the Instead of one low-energy conformation dominating the L650T͞ L650T͞AMPA complex to the corresponding complex with AMPA free energy spectrum, which would be the conformation glutamate or quisqualate, we determined the cocrystal structure with full domain closure in the case of the WT receptor, there of the L650T͞quisqualate species under crystallization condi- are at least two conformations, one with Ϸ11° and a second with tions that were essentially identical to those used to grow the AS Ϸ21° of domain closure. The multiple conformations of the and Zn crystal forms of L650T͞AMPA. We focused on the ligand-binding core result in submaximal AMPA currents, be- L650T͞quisqualate complex because it more readily produced cause the conformers with Ͻ21° of domain closure do not allow diffraction-quality crystals in comparison with the glutamate the ligand-binding core to do as much work on the ion channel complex. In addition, structural analysis of the WT GluR2 in comparison with the conformers that possess full domain S1S2J͞quisqualate complex has shown that quisqualate is a bona closure. fide isostere of glutamate (30) and in the present study we found that quisqualate acted as a full agonist on GluR2 L650T mutant Linker Separation Is Correlated to Channel Activation. Illustrated in receptors. All of the refined L650T͞quisqualate structures show Fig. 5 is a graph showing the correlation between linker separation and Imax values for the WT and L650T variants of the GluR2 receptor with full and partial agonists, as well as the apo- and antagonist-bound forms. As the extent of linker separation increases from Ϸ34.3 Å in the WT͞kainate dimer to Ϸ37.2 Å in the WT AMPA, quisqualate, and glutamate dimer (18), the

Fig. 5. Correlation between linker separation and agonist efficacy. The values for relative agonist efficacy were determined for L483Y receptors (F) Fig. 6. Mechanism of agonist binding and domain closure. (A) The binding and WT receptors in the presence of 100 ␮M cyclothiazide (■) and are listed site of the open-cleft, closed-channel state (Apo S1S2J, protomer A). (B) The in Table 1. Apo and 6,7-dinitroquinoxaline-2,3-dione (DNQX) are assumed to possible first step in agonist binding as observed in molecule B of the L650T͞ have an efficacy of zero, and the other values are scaled to glutamate, which AMPA(AS) structure. We suggest that this semiclosed cleft conformation is set to one. Linker separation is defined as the distance between Gly C␣ atoms represents the agonist-bound, closed-channel state. (C) The closed-cleft, from the artificial Gly-Thr linker joining S1 and S2. The structure correspond- open-channel conformation as observed in the WT S1S2J͞AMPA binding cleft ing to each point is indicated. The L650T͞AMPA(Zn) structure is indicated with (protomer A). In B and C, water molecules are shown as green spheres, AMPA an ϫ. The glutamate (Glu) and quisqualate (Quis) points are all clustered is drawn in magenta, and hydrogen bonds are depicted by black dashed lines. around an efficacy of 1.0. The agonist-bound data points were fit with a linear The degrees of domain closure relative to the Apo conformation are indicated equation that yields a correlation coefficient of 0.893. KA, kainate. below each structure.

5740 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1037393100 Armstrong et al. Downloaded by guest on September 29, 2021 ͞ relative Imax values also increase from 0.02 to 1.0. A simple linear L650T AMPA structure, the isoxazole ring of AMPA initially fit to the points associated with the full and partial agonists yields contacts the hydroxyl of Ser-654 and the ligand-binding core a striking correlation, thus strengthening the concept that do- closes by Ϸ11°. After the formation of this initial complex, main closure and linker separation are coupled to the activation domain 2 moves toward domain 1, increasing the extent of of the ion channel and that agonists that yield maximal domain domain closure to Ϸ21°, allowing for the formation of additional closure and linker separation produce maximal activation of the direct receptor contacts with AMPA, and trapping the agonist in ion channel. the binding pocket (25). We suggest that this final rearrangement of domain 2 is the primary conformational change induced by Mechanism of Agonist Binding and Domain Closure. The partially AMPA that is coupled to the gating or opening of the ion closed conformation seen in subunit B of the L650T͞AMPA channel. (ammonium sulfate form) complex fortuitously affords a view of Conclusions a putative agonist-bound, closed-channel state. AMPA is bound primarily to residues in domain 1, making contacts to domain 1 Understanding the function, mechanism, and symmetry of al- that are essentially identical to those made upon full domain losteric proteins necessarily requires structural information. In closure. However, AMPA only makes one direct and three the case of ligand-gated ion channels there are, at the present water-mediated interactions to domain 2, in comparison with the time, no atomic resolution structures of a ligand-gated ion three direct and three water-mediated interactions made upon channel in apo- and agonist-bound states. Our structural studies full domain closure. The partially closed L650T͞AMPA subunit on the GluR2 ligand-binding core, while not including the ion channel domain, nevertheless demonstrate that the agonist- structure, in conjunction with previously determined structures, binding region can adopt a range of agonist-dependent confor- allows us to piece together a mechanism for agonist binding to mations, thus suggesting that AMPA receptors are best the resting, closed-channel state of the receptor and for the described by an induced-fit, multistate model. The extent to subsequent conformational rearrangements in the ligand- which other ligand-gated ion channels, such as cyclic-nucleotide- binding core. Shown in Fig. 6 is an illustration of this process, gated channels and members of the acetylcholine receptor family which is also seen in Movie 1, which is published as supporting (31), conform to a two-state versus a multistate model must information on the PNAS web site. await further structural and functional investigations. The proposed mechanism for AMPA binding and ensuing conformational changes is as follows. In the resting, inactive Joe Lidestri is gratefully acknowledged for the direction and mainte- state, the ligand-binding cleft is open, the ion channel is closed, nance of the x-ray laboratory at Columbia University. Craig Ogata is and the carboxylate of Glu-705 interacts with the hydroxyl group thanked for assistance with data collection at X4A at the National of Thr-655 at the base of helix F and the amino group of Lys-730 Synchrotron Light Source, and Carla Glasser provided valuable assis- (14). Initially, AMPA binds to the ligand-binding core via tance with the physiology experiments. We thank Cinque Soto for interactions with preorganized residues on domain 1 and the assembling the S1S2J L650T construct. This work was supported by the National Institutes of Health (to M.M. and E.G.), the National Alliance carboxylate of Glu-705 moves from the base of helix F to bind for Research on Schizophrenia and Depression (to E.G.), and the Jane to the amino group of AMPA, thus opening up the anion binding Coffin Childs Memorial Fund for Medical Research (to N.A.). E.G. is an site at the base of helix F for the isoxazole ring of AMPA or the Assistant Investigator of the Howard Hughes Medical Institute and a ␥-carboxylate of glutamate. As revealed in molecule B of the Klingenstein Research Fellow.

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Armstrong et al. PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 5741 Downloaded by guest on September 29, 2021