Research Article

Structure of a Class C GPCR Metabotropic Glutamate Receptor 1 Bound to an Allosteric Modulator

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Science  04 Apr 2014:
Vol. 344, Issue 6179, pp. 58-64
DOI: 10.1126/science.1249489

Completing the Set

G protein–coupled receptors (GPCRs) are membrane proteins that transduce extracellular signals to activate diverse signaling pathways. Significant insight into GPCR function has come from structures of three of four classes of GPCRs—A, B, and Frizzled. Wu et al. (p. 58, published online 6 March) complete the picture by reporting the structure of metabotropic glutamate receptor 1, a class C GPCR. The structure shows differences in the seven-transmembrane (7TM) domain between class C and other classes; however, the overall fold is preserved. Class C GPCRs are known to form dimers through their extracellular domains; however, the structure suggests additional interactions between the 7TM domains mediated by cholesterol.

Abstract

The excitatory neurotransmitter glutamate induces modulatory actions via the metabotropic glutamate receptors (mGlus), which are class C G protein–coupled receptors (GPCRs). We determined the structure of the human mGlu1 receptor seven-transmembrane (7TM) domain bound to a negative allosteric modulator, FITM, at a resolution of 2.8 angstroms. The modulator binding site partially overlaps with the orthosteric binding sites of class A GPCRs but is more restricted than most other GPCRs. We observed a parallel 7TM dimer mediated by cholesterols, which suggests that signaling initiated by glutamate’s interaction with the extracellular domain might be mediated via 7TM interactions within the full-length receptor dimer. A combination of crystallography, structure-activity relationships, mutagenesis, and full-length dimer modeling provides insights about the allosteric modulation and activation mechanism of class C GPCRs.

The human G protein–coupled receptor (GPCR) superfamily comprises more than 800 seven-transmembrane (7TM) receptors that can be divided into four classes according to their sequence homology: class A, B, C, and F (Frizzled) (1). Class C GPCRs play important roles in many physiological processes such as synaptic transmission, taste sensation, and calcium homeostasis; they include metabotropic glutamate receptors (mGlus), γ-aminobutyric acid B (GABAB) receptors, calcium-sensing (CaS) receptors, and taste 1 (TAS1) receptors, as well as a few orphan receptors. A distinguishing feature of class C GPCRs is constitutive homo- or heterodimerization mediated by a large N-terminal extracellular domain (ECD) (Fig. 1A). The ECDs within homodimeric receptors (mGlu and CaS) are cross-linked via an intermolecular disulfide bond. The heterodimeric receptors (GABAB and TAS1) are not covalently linked, but their heterodimerization is required for trafficking to the cell surface and signaling (2). The ECD of class C GPCRs consists of a Venus flytrap domain (VFD), which contains the orthosteric binding site for native ligands (Fig. 1A), and a cysteine-rich domain (CRD), except for GABAB receptors. The CRD, which mediates the communication between ECD and 7TM domains, is stabilized by disulfide bridges, one of which connects the CRD and VFD (3).

Fig. 1 Overall structure of the mGlu1 TM domain.

(A) Cartoon models for structure and endogenous ligand recognition in different GPCR classes. For class A, in most cases, the endogenous ligand (green) is recognized by an orthosteric site in the 7TM domain. For class B, the endogenous peptide ligand (orange) binds to both ECD and 7TM domains. For class C, the endogenous small-molecule ligands (yellow circle) are recognized by orthosteric sites in the VFDs. For class F, lipoprotein WNT (magenta) binds the CRD domain of Frizzled receptors. (B) The mGlu1 7TM domain that crystallized as a parallel dimer is shown in cyan cartoon. Cholesterols mediating the dimer interface are shown as green carbons. (C) Side chains of the FITM binding pocket residues are shown as white carbons. Hydrogen bond interaction between the NAM and Thr8157.38 is shown as a dashed line. The Cα carbon of Gly7615.48 is shown as a green ball. In (B) and (C), the ligand FITM is shown as yellow carbons. Amino acid abbreviations: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

The mGlu family was the first group of class C GPCRs to be cloned (4, 5). Comprising eight members, the mGlu family can be separated into three subgroups (6)—group I (mGlu1 and mGlu5), group II (mGlu2 and mGlu3), and group III (mGlu4, mGlu6, mGlu7, and mGlu8)—on the basis of sequence homology, G protein–coupling profile, and pharmacology (7). Group I mGlus are predominantly coupled to Gq/11 and activate phospholipase Cβ, which hydrolyzes phosphoinositides into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, thereby inducing intracellular calcium mobilization and activating protein kinase C (PKC).

The group I mGlus, mGlu1 and mGlu5, are considered promising therapeutic targets to treat diseases including cancer, chronic pain, schizophrenia, Alzheimer’s disease, anxiety, and autism (7, 8). However, the development of subtype-selective small-molecule ligands that might serve as drug candidates for these receptors has been hampered by the conservation of the orthosteric (glutamate) binding site (Fig. 1A). This problem can be overcome by using allosteric modulators that act at alternative binding sites; these compounds bind predominantly within the 7TM domain of the class C receptors. Allosteric modulators can alter the affinity or efficacy of native ligands in positive, negative, and neutral ways, demonstrating a spectrum of activity that cannot be achieved by orthosteric ligands alone.

Here, we report the crystal structure of the human mGlu1 7TM domain bound to a negative allosteric modulator (NAM), 4-fluoro-N-(4-(6-(isopropylamino)pyrimidin-4-yl)thiazol-2-yl)-N-methylbenzamide (FITM) (9), at 2.8 Å resolution (table S1) (10). This structure provides a three-dimensional framework for understanding the molecular recognition and facilitating the discovery of allosteric modulators for the mGlu family and other class C GPCRs. It also complements crystallographic studies of the transmembrane domain structures of class A (11, 12), B (13, 14), and F (15) GPCRs and extends the knowledge base upon which to study the diversity and evolution of the GPCR superfamily.

Overall Structure of the mGlu1 7TM Domain

The human mGlu1 7TM domain (residues 581 to 860) (fig. S1), complexed with FITM, was crystallized by the lipidic cubic phase method using the thermostabilized apocytochrome b562RIL (BRIL) N-terminal fusion strategy (10). A series of in vitro pharmacological studies were performed to verify that this truncated construct binds FITM and is functional in G protein coupling (figs. S2 and S3). The structure was solved using a 4.0 Å single-wavelength anomalous dispersion (SAD) data set collected from a single crystal soaked with tantalum bromide cluster; the resolution was then improved to 2.8 Å by means of native data collected from 14 crystals (table S1 and fig. S4).

The mGlu1 7TM domain forms a parallel dimer in each asymmetric unit, with a dimer interface mediated mainly through helix I (Fig. 1B; see also figs. S5 and S6). We observed six well-resolved cholesterol molecules packed against hydrophobic residues on the extracellular side of helices I and II, mediating the dimer formation. The extracellular loop (ECL) 2 adopts a β-hairpin conformation, pointing to the extracellular space, which has also been observed in many peptide class A GPCRs (16, 17). This β hairpin is connected to the top of helix III through a disulfide bond (C657-C746) that is conserved through all classes of GPCRs. The mGlu1 NAM, FITM, binds within a pocket formed by the 7TM bundle close to the extracellular side (Fig. 1, B and C), a region that partially overlaps with the orthosteric binding sites observed for class A GPCRs (11). The intracellular loop (ICL) 1 forms an ordered helical turn; a large part of ICL2 (residues 688 to 695 in molecule A, residues 689 to 693 in molecule B) is missing in the structure because of the long and presumably flexible nature of this loop. ICL3 is well resolved and forms a short link connecting the intracellular ends of helices V and VI. In addition, we did not observe helix VIII, reported in most class A GPCR structures as well as in classes B and F. Instead, electron densities for C-terminal residues (844 to 860 in molecule A, 847 to 860 in molecule B) are missing in the mGlu1 structure, indicating that this region can be disordered.

Major Structural Differences with Other GPCR Classes

Superposition of 7TM domains between mGlu1 and GPCRs of different classes (fig. S7) reveals that, despite the lack of sequence conservation (<15% identical residues) or common functional motifs (figs. S8 and S9), the overall fold is preserved across the whole GPCR superfamily (root mean square deviation <3.5 Å for 7TM regions). On the basis of structural superposition, we generated a structure-based alignment in the 7TM domain with class A GPCRs and transplanted the class A Ballesteros-Weinstein (B&W) numbering (18) to class C GPCRs (figs. S8 and S9) (19).

Differences, however, were observed in the 7TM helices between class C and other classes, including distinct distribution patterns of proline-induced kinks in the helical backbone. Instead of having conserved prolines in the X.50 position of helices V, VI, and VII (which induce kinks as observed in class A), residues at the 5.50, 6.50, and 7.50 positions in mGlu1 are all nonproline residues (fig. S8). Notably, Pro8337.56 (20) at the intracellular end of helix VII in mGlu1 induces a kink, resulting in an outward orientation of the C-terminal part of this helix (fig. S7B). In contrast, the proline conserved in the class A Asn-Pro7.50-x-x-Tyr motif is on the opposite side of helix VII and induces an inward kink (fig. S7B).

Helices I to IV of mGlu1 overlay relatively well with other GPCR structures, whereas helices V to VII demonstrate more obvious differences. Relative to class A and B receptors (Fig. 2, A and B), helix V of mGlu1 is shifted inward to the center of the 7TM bundle. Additionally, the extracellular end of helix VII is shifted inward relative to all other classes. These shifted helices, together with ECL2, restrict access to the NAM-binding cavity (Fig. 2, D and E). The recently solved class F smoothened receptor (15) also has a narrow cavity embedded in the extracellular half of its 7TM bundle, resulting partly from the inward positioning of helix V, but its ECL2 β hairpin is located inside the 7TM bundle and forces an outward shift of helix VII as compared to mGlu1 (Fig. 2C). This more restricted 7TM cavity in class C receptors is consistent with interactions of known native ligands with the ECD rather than the 7TM domain.

Fig. 2 7TM domain comparison of mGlu1 with GPCRs of other classes.

(A to C) Extracellular view of superpositions between mGlu1 shown in cyan and (A) class A GPCR shown in salmon, (B) class B GPCR (CRFR1 and glucagon receptor; PDB IDs 4K5Y and 4L6R, respectively) shown in gray, and (C) class F GPCR (smoothened receptor; PDB ID 4JKV) shown in purple. In (A) to (C), shifts of helix V or VII in mGlu1 relative to other classes are indicated by arrows. (D) Extracellular view of the surface presentation of the structure, showing the narrow entrance (highlighted by red line) to the allosteric ligand-binding cavity in the center. (E) A cut-through surface side view of the ligand-binding cavity. The arrow indicates the extracellular entrance to the allosteric ligand-binding cavity. In the surface presentations in (D) and (E), nonpolar residues are shown in gray, hydrogen bond acceptors are in red, and hydrogen bond donors are in blue. PDB IDs of class A GPCRs structures used in (A): 1U19, 2RH1, 2YCW, 3RZE, 3PBL, 3UON, 4DAJ, 3EML, 3V2W, 3ODU, 4DJH, 4EA3, 4DKL, 4EJ4, 3VW7, and 4GRV.

The FITM Binding Pocket

Analogous to the orthosteric site for many family A GPCRs, the binding pocket for the ligand FITM is defined by residues on ECL2 and helices II, III, V, VI, and VII (Fig. 1C and fig. S10). ECL2 forms a lid on the top of the ligand-binding cavity, leaving a small opening through which the pocket is accessible from the extracellular side (Fig. 2, D and E). The ligand, FITM, fits tightly into the long and narrow pocket. Most of the ligand-receptor interactions are hydrophobic, with the exception of the contacts of the pyrimidine-amine group with the Thr8157.38 side chain. Substitution of Thr8157.38 with methionine or alanine reduces the affinity and potency of FITM (Fig. 3B, fig. S11F, and table S2) and of other mGlu1 NAMs from different scaffolds (2123). The p-fluorophenyl moiety of the ligand points to the bottom of the pocket, making contacts with Trp7986.48, a residue that is conserved among mGlus as well as in many class A receptors. However, unlike the conformation of the Trp6.48 side chain observed in most class A GPCRs, which points into the center of the helical bundle, Trp7986.48 in the structure of FITM-bound mGlu1 points outward. This conformation of the bulky indole group is accommodated by Gly7615.48 on helix V, which has no side chain (Fig. 1C). However, in mGlus other than mGlu5, residues at position 5.48 have relatively large side chains and Trp6.48 may adopt a different conformation.

Fig. 3 Critical FITM-receptor interactions are revealed by mutations and structure-activity relationships.

(A to C) FITM is a full NAM of the wild-type (WT) full-length human mGlu1 receptor (A); the affinity of FITM and the degree of negative cooperativity with glutamate are reduced in Thr8157.38Met (B) and Pro7565.43Ser (C) mutants (table S3). Error bars denote SEM. (D) Structures of FITM and FITM-related NAMs used for study. (E to G) Binding pose of FITM (yellow carbons; mean half-maximal inhibitory concentration IC50 = 5 nM) in comparison with lower-potency analogs: (E) compound 17 (green carbons; IC50 = 10 nM) and compound 14 (orange carbons; IC50 = 230 nM), (F) compound 28 (gray carbons; IC50 = 77 nM) and (G) compound 22 (purple carbons; IC50 = 2 μM). Per-residue binding energy ddG is predicted by Rosetta and shown as Rosetta Energy Units (REU) (37). In (E) to (G), side-chain rotamers from the top 1% of key amino acids are depicted in sticks and colored corresponding to their respective docked ligand, with the exception of those from the crystal structure shown in cyan; the dashed lines indicate hydrogen bond interactions between the receptor and the ligands.

Determinants of Subtype Selectivity Within the Common Allosteric Site

Previous mutagenesis studies have proposed at least one common allosteric site for the mGlu family within the 7TM domain. The mGlu1 binding pocket for FITM (Fig. 1C) largely corresponds to mutagenic data for the common allosteric site in mGlus and likely extends to other class C GPCRs (see table S3). Despite the evidence that binding of various chemotypes of class C GPCR allosteric modulators involve similar residue positions, many mGlu modulators display a high degree of subtype selectivity, including FITM, which shows high affinity (equilibrium dissociation constant Ki = 2.5 nM; fig. S2) and selectivity for mGlu1 over mGlu5 (fig. S12) (9). Examination of the contact residues in the binding pocket reveals only four residues of mGlu1 that differ from mGlu5: Val6643.32, Ser6683.36, Thr8157.38, and Ala8187.41, all of which have previously been implicated in subtype selectivity by mutagenesis-based studies (2426). Therefore, we mutated these four residues to their corresponding amino acid in mGlu5 (fig. S11 and table S2) and compared FITM-mediated antagonism of the mutant receptors to the wild-type full-length human mGlu1 (Fig. 3A). Methionine substitution of Thr8157.38 (Fig. 3B) had the most profound effect, reducing FITM affinity by a factor of ~6 and decreasing negative cooperativity with glutamate (table S2). Thus, Thr8157.38 is a key selectivity determinant for FITM, consistent with the observed polar interaction between Thr8157.38 and the ligand in the structure.

In addition, we assessed mutations known to influence the allosteric modulation of other mGlu subtypes that had not previously been explored in mGlu1. Thr7946.44Ala and Ser8227.45Ala mutations had no effect on FITM, whereas Pro7565.43Ser reduced FITM affinity (by a factor of ~3) as well as negative cooperativity (Fig. 3C and table S2). Location of Pro7565.43 in the ligand-binding pocket suggests that a Pro7565.43Ser mutation may induce conformational changes in the backbone, altering the shape of the binding pocket in relation to the thiazole core of FITM.

Multiple mGlu5 modulator scaffolds are known to be sensitive to mutations of two nonconserved residues, Ser6.39 and Ala7.46 (23, 25, 2731); neither is observed here as contributing to the FITM-binding pocket. However, both of these residues contribute to a small pocket separated from the FITM pocket by the Tyr6723.40 side chain. Given that Ser3.36 in mGlu1 is replaced by Pro3.36 in mGlu5, it is conceivable that the proline-induced kink in helix III particular to mGlu5 may change the shape of the pocket, making Ser6.39 and Ala7.46 of mGlu5 accessible to ligands.

To further improve our understanding of the critical ligand-receptor interactions for FITM binding within the pocket, we docked a selection of FITM analogs (Fig. 3, D to G, and fig. S13) (9) into the crystal structure. Redocking FITM (fig. S13, A to C) and analyzing the binding energy contribution per residue (fig. S13D) revealed that Thr8157.38 forms an energetically favorable hydrogen bond with FITM (fig. S13, C and D). Compound 17 lacks not only the hydrogen bond with Thr8157.38, but also a nonpolar interaction with Leu6482.60 (Fig. 3, D and E); however, a potential hydrogen bond with Gln6603.28, which is not observed in FITM binding (fig. S11A and table S2), may compensate for this loss and account for the retained activity at 10 nM. The 3-pyridyl analog (compound 14; Fig. 3, D and E) lacks this potential interaction with Gln6603.28, accounting for its further decreased potency (230 nM). Compound 28 exhibits lower potency (by a factor of ~10) and differs from FITM by the introduction of a methyl group to the amine on the pyrimidine ring. Docking compound 28 reveals a major energy penalty that arises from the loss of a polar interaction and the introduction of steric clash with Thr8157.38 (Fig. 3, D and F). Compound 28 also lacks a polar interaction with Gln6603.28 and requires movement of Thr8157.38 and Tyr8056.55 to accommodate the methyl group (Fig. 3F). Compound 22 (Fig. 3, D and G), which contains an isopropyl group on the amide linker, requires movement of two residues in helix V (Pro7565.43 and Leu7575.44) to fit in the pocket, and also lacks hydrogen-bonding capacity with either Thr8157.38 or Gln6603.28, accounting for its reduced (micromolar) potency. Collectively, by comparing the binding of FITM with those of other less active or inactive compounds, we attribute the superior potency observed for FITM to the polar interaction between Thr8157.38 and the amine derivative on the 5′ position of the pyrimidine ring, as well as the perfect fit of the ligand shape within the narrow binding pocket.

NAM-Bound mGlu1 Is in an Inactive State

In the NAM-bound structure of mGlu1, the intracellular site responsible for G protein interaction is in a conformation similar to the inactive conformation observed in class A GPCRs (Fig. 4, A to C). One of the interactions apparently stabilizing this conformation is a salt bridge between the Lys6783.46 side chain at the intracellular end of helix III and the Glu7836.33 side chain at the intracellular end of helix VI (Fig. 4, D and E); both residues are well conserved in all class C receptors. In class A GPCRs, a similar interaction called an “ionic lock” is observed between the conserved Arg3.50 of the Asp(Glu)-Arg3.50-Tyr motif and an acidic residue in the 6.30 position. The “ionic lock” plays a role in stabilizing the receptor’s inactive state by restricting the activation-related outward movement of helix VI. In addition to the salt bridge between Lys6783.46 and Glu7836.33, Ser626ICL1—a residue conserved in most class C GPCRs—also participates in this interaction network. Ser625ICL1 and Asn780ICL3 form a hydrogen bond, stabilizing the interaction between ICL1 and ICL3 and further occluding the G protein–binding site (Fig. 4, D and E). Thus, polar interactions within the intracellular crevice may be involved in the regulation of G protein binding and receptor activation in both class A and class C GPCRs, although through distinct residue positions.

Fig. 4 The intracellular crevice in NAM-bound mGlu1 adopts a closed conformation.

(A) Cartoon demonstrating agonist-triggered opening of the intracellular cavity for G protein binding. (B and C) Side view (B) and intracellular view (C) of the superposition of mGlu1 (cyan) with inactive state of β2-adrenergic receptor shown in yellow (PDB ID 2RH1) and a fully active G protein–coupled state of β2-adrenergic receptor shown in orange (PDB ID 3SN6). Red arrows in (B) and (C) indicate movement of the intracellular end of helix VI, highlighted in red dashed circle in (B), during activation of β2-adrenergic receptor. (D and E) Side view (D) and intracellular view (E) of the mGlu1 receptor; side chains of residues involved in a hydrogen bond network that stabilize the receptor in an inactive conformation are shown as white carbons.

Communication Between ECD and 7TM Domains

In the mGlu receptor family, as well as in other class C GPCRs, a signal is initiated by the native ligand binding to the ECD, which induces conformational changes in the ECD. In our structure, the linker region (Ile581-Glu592) between the ECD and 7TM domain is resolved. The linker forms strong interactions with the ECL2 β sheet through main-chain and side-chain hydrogen bonds (Fig. 5A). ECL2 is connected by a covalent disulfide bond to the top of helix III, known to be important in triggering activation in class A GPCRs (32). This observation raises the possibility that this interaction network might contribute to the communication between the ECD and the 7TM domain during receptor activation. In addition, part of the linker residues (e.g., Trp588, a residue conserved in all mGlus) insert into the lipid bilayer, where they form extensive contacts with cholesterol molecules that mediate the observed dimerization of the 7TM domain (fig. S6). These interactions suggest a potential role of dimerization and/or lipid components in the coupling between the ECD and 7TM domain during the activation process.

Fig. 5 A full-length mGlu1 dimer model with highlighted details of interactions between ECL2 and the 7TM-CRD linker.

(A) Shown in cyan is the extracellular part of the mGlu1 7TM. ECL2 residues (Met731 to Ile745) are shown as white carbons; the linker region residues (Ile581 to Glu587) are shown as yellow carbons. Hydrogen bond interactions between ECL2 and the linker region are shown as dashed lines. (B) Full-length model of mGlu1 with the VFD in the Acc (active closed-closed) state. VFD, CRD, and 7TM domains are colored in slate, firebrick, and cyan, respectively. The current model probably does not capture the specific conformation and interaction between the CRD and 7TM domains, and a more tightly packed domain interaction is very likely. This model is presented to generate discussion and to show the general features of the VFD, CRD, and 7TM domains.

ECDs of class C GPCRs mediate receptor homo- and heterodimerization (2). Several dimeric structures of mGlu receptor VFDs have been solved in different conformations: putative active (A) or resting (R) states defined by the relative orientation between the VFD protomers, as well as closed (c) or open (o) states defined by the conformation of each VFD (33). Comparing different conformations, the distance between the C-terminal ends of the ECDs within a dimer changes markedly (3). In our crystal structure of the 7TM domain, we observed a parallel dimer mediated by interactions of helix I and cholesterols. In this dimer conformation, the distance between the N-terminal linkers that are attached to the C termini of ECDs is ~20 Å. If this is a conformation that can be adopted by the full-length receptor dimer, the CRDs of each protomer should also be in close proximity. Disulfide bond cross-linking experiments suggested that the CRDs of each protomer may form close contact in an activated receptor dimer (34). Although our structure is solved in complex with a NAM and the 7TM domain appears to be in an inactive state, there is evidence supporting the existence of a glutamate-bound but signaling-incapable state in the full-length mGlu dimer (35). Moreover, there is evidence that cholesterol can positively modulate glutamate responses by recruiting mGlus to lipid rafts (31, 36), consistent with the observation that the close proximity of the N terminus of the 7TM domain results from a dimer conformation mediated by multiple cholesterol molecules.

To test the possibility of fitting the existing ECD structures into our observed 7TM dimer conformation, we created a full-length dimer model in which the VFD adopts an Acc (active closed-closed) conformation, as this conformation has the closest distance (~50 Å) between the C termini of the ECDs (3) (Fig. 5B). A 20° rotation was applied to the CRD, coupled with a conformational change in the Gln513-Val523 loop region that reduces this distance to 20 Å, fulfilling the CRD interface proposed in the cysteine mutant study (34), as well as matching the distance of the 7TM domain N termini observed in the crystallographic dimer. This model might represent a glutamate-bound but signaling-incapable conformation of mGlu1. Although this model is consistent with the currently available experimental data, we acknowledge that it is only one of several possible explanations for the biological role of the 7TM domain dimer we observed. We further acknowledge that the 7TM domain dimer conformation might vary in different states of the receptor and may be modulated by several factors in biological systems, such as membrane lipid content or other protein-protein interactions.

The mGlu1 7TM structure presented here uncovers atomic details of the class C GPCR transmembrane domain, providing a missing link in our structural understanding of the GPCR superfamily. As noted for the recently solved class B and class F GPCR structures, and now for class C, despite a lack of sequence and motif conservation, the architecture of the 7TM bundle is generally preserved. Furthermore, although class C GPCRs are known to form obligate dimers via the ECDs, the observed 7TM dimer suggests additional points of communication between protomers, mediated by multiple cholesterol molecules and direct protein-protein interactions. Moreover, as a robust structural template, the mGlu1 7TM domain structure will likely provide insights into pharmacology of small-molecule allosteric modulators for class C GPCRs.

Supplementary Materials

www.sciencemag.org/content/344/6179/58/suppl/DC1

Materials and Methods

Tables S1 to S3

Figs. S1 to S13

References (3871)

References and Notes

  1. See supplementary materials on Science Online.
  2. In each helix, the following residues are assigned number 50: T6071.50, I6382.50, I6823.50, I7144.50, L7635.50, A8006.50, and L8277.50. The numbering of other residues in each helix is counted relative to the X.50 position according to the B&W numbering system.
  3. Superscripts refer to the B&W numbering for class A GPCRs and were transplanted to class C GPCRs on the basis of structural superposition.
  4. Acknowledgments: Supported by National Institute of General Medical Sciences (NIGMS) PSI:Biology grant U54 GM094618 for biological studies and structure production (target GPCR-68) (V.K., V.C., and R.C.S.); NIH Common Fund in Structural Biology grant P50 GM073197 for technology development (V.C. and R.C.S.); NIH grants R01 NS031373 (P.J.C.), R01 MH062646 (P.J.C.), and R21 NS078262 (C.M.N.); a basic research grant from the International Rett Syndrome Foundation (C.M.N.); an NHMRC (Australia) Overseas Biomedical postdoctoral fellowship (K.J.G.); and a NARSAD Maltz Young Investigator award (K.J.G.). Work in the Meiler laboratory on computational modeling of membrane proteins and their ligand interactions is supported by NIH grants R01 MH090192, R01 GM080403, R01 GM099842, and R01 DK097376 and by NSF grant CHE 1305874. We thank K. Emmitte and P. Garcia for the synthesis of RO0711401 and FITM; J. Velasquez for help on molecular biology; T. Trinh and M. Chu for help on baculovirus expression; K. Kadyshevskaya for assistance with figure preparation; A. Walker for assistance with manuscript preparation; Q. Xu for help on SHARP density modification; and J. Smith, R. Fischetti, and N. Sanishvili for assistance in development and use of the minibeam and beamtime at GM/CA-CAT beamline 23-ID at the Advanced Photon Source, which is supported by National Cancer Institute grant Y1-CO-1020 and NIGMS grant Y1-GM-1104. Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4OR2.
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