The ten mammalian Frizzleds (FZD1–10) belong to the class F of G protein-coupled receptors (GPCRs) and mediate WNT signaling through interaction with transducer proteins including Dishevelled (DVL) or heterotrimeric G proteins.1 Their involvement in human disease has put FZDs at the forefront of drug targets, especially anti-cancer therapy.2 However, no drugs have been developed for efficient pharmacological modulation of FZDs, partially owing to the limited understanding of FZD structure and activation mechanisms.1,3 Among class F, FZD7 is intensively pursued due to its relevance in various tumor models, particularly in intestinal cancers.4 Detailed structures of the receptor complexes would allow for structure-guided discovery of new drug candidates. FZD1–10 share structural similarity with the related class F member Smoothened (SMO), which mediates Hedgehog signaling and is a validated target for cancer therapy.2 In an effort to understand the structural basis of FZD activation and transducer interaction, we solved the structure of human FZD7 in complex with heterotrimeric mini Gs (mGs).5
Based on the evidence that FZD7 interacts with Gs to mediate muscle hypertrophy,6,7 we assessed its ability to activate heterotrimeric Gs independently of WNT stimulation. Co-expression of FZD7 with a bioluminescence resonance energy transfer (BRET)-based Gs biosensor,8 reporting the rearrangement or dissociation of Gαs and Gβγ following receptor engagement and G protein activation, revealed that FZD7 exhibits constitutive activity similar to the class A β2-adrenoceptor (Fig. 1a; Supplementary information, Fig. S1a, b). Using an analogous assay that measures activity-dependent Gαs translocation (Supplementary information, Fig. S1c), we found that the constitutive activity of FZD7 correlates with increased receptor expression (Supplementary information, Fig. S1d, e). Given the robust constitutive activity of FZD7 towards Gs, we reconstituted purified, full-length human FZD7, heterotrimeric mGs and Nanobody35 (Nb35), which stabilizes the nucleotide-free Gαs and Gβ subunits,9 in the absence of ligand and obtained pure complexes following size exclusion chromatography (Supplementary information, Fig. S2). The final complex was composed of FZD7, mGαs, Gβ, Gγ and Nb35, which could be clearly identified by 2D classification (Fig. 1b; Supplementary information, Fig. S2d). We used single-particle cryo-EM analysis to determine the 3D structure of this complex. After several rounds of classification and auto-refinement, the resolution of the final structure reached 3.2 Å allowing us to build an atomic model based on the density map (Fig. 1c; Supplementary information, Figs. S3–S5, Table S1).
In accordance with the functional evidence for constitutive activity, the FZD7–mGs complex structure provides the structural basis for ligand-independent G protein coupling (Fig. 1c). The interface between FZD7 and mGs is dominated by the distal C-terminal segment of the α5-helix in mGαs (Fig. 1d, e). The C-terminal leucine residues (L393H5.25, L394H5.26; superscripts refer to the residue position in the common Gα numbering scheme for G proteins/GPCRdb) are inserted into the helical bundle of the receptor. L393H5.25 and L394H5.26 establish extensive interactions with FZD7 residues yielding a locally converged network that stabilizes the complex (Fig. 1e). The terminal carboxyl group of L394H5.26 in mGαs forms an ionic bond with K4666.28, and residues R281ICL1, K5528.49 and R4706.32 of FZD7 are located in close proximity (superscript numbers refer to the Ballesteros and Weinstein numbering system). Y391H5.23 forms a hydrogen bond with the backbone of W369ICL2. Residues I4505.72, I4535.75 and M4545.76 of FZD7 form a hydrophobic cleft accommodating L388H5.20. Furthermore, R385H5.17 forms an ionic bond with D457 in ICL3, further strengthening the interaction between the α5-helix and FZD7. In summary, the recognition of Gαs by FZD7 is primarily governed by a network of hydrogen bonding and electrostatic interactions contributed from the C-terminal segment of the α5-helix (D381H5.13-L394H5.26), among which, interactions with L394H5.26 lock the α5-helix tail in an uncoiled, elongated conformation (Fig. 1e).
The placement of the α5-helix of mGαs in the core of FZD7 stabilizes an open FZD7 conformation. We compared the FZD7–mGs structure with the available inactive-state FZD4 crystal structure (PDB: 6BD4) and the inactive-state FZD5 cryo-EM structure (PDB: 6WW2) and observed a clear outward bending of TM6 and an inward shift of TM5 at the cytoplasmic side (Fig. 1f–h)—a conformational change characteristic of active-state class A and B GPCRs. This helical rearrangement is achieved through interaction of TM6 and TM5 with mGs and opening of the molecular switch between TM6 and TM7 (R6.32/W7.55; Fig. 1i).7 Comparing inactive FZD4 with FZD7–mGs reveals that the extracellular portion of TM6 of FZD7 extends above the surface of the lipid bilayer at an angle of 45° (Supplementary information, Figs. S6, S7), similar to what we have predicted in previous models10 and in contrast to the almost 90° bending in the FZD4 structure.11 Moreover, conserved cysteines within the hinge domain form disulfide bonds to both stabilize its structure and to link it with ECL1 (C210–C230; C234–C315ECL1) (Supplementary information, Fig. S6).12
To better understand the activation mechanism of FZD7 and G protein coupling to class F receptors, we compared the FZD7–mGs structure with the agonist (24(S), 25-epoxycholesterol)-bound structure of SMO–Gi13 (PDB: 6OT0). The helical arrangement at the upper portion of the FZD7 transmembrane core is more compact, presumably due to the absence of ligand (Supplementary information, Fig. S7). At the lower portion of TM6, substantially distinct conformations are observed between the SMO–Gi and FZD7–mGs structures. Most strikingly, TM6 in SMO–Gi undergoes a parallel outward movement compared to inactive SMO, whereas TM6 in the FZD7–mGs complex accomplishes a similar displacement of the cytoplasmic portion through a kink in the helix (Fig. 1h). The ionic interactions between TM6, ICL3 and the α5-helix of mGαs (K4666.28–L394H5.26 and D457–R385H5.17) are likely to be the main contributors in maintaining this kink. In addition, Y4786.40 forms π–π interaction with W3543.43 to further maintain the bent TM6 conformation (Supplementary information, Fig. S7).
While the most evident structural rearrangements relate to TM6, additional positional shifts of TM2, TM3, TM4 and TM5 in the FZD7–mGs complex are observed when compared to the SMO–Gi complex. These four helices constitute a more compact bundle in the FZD7–mGs structure, partially stabilized by a network of π interactions (Supplementary information, Fig. S7e). In a cooperative manner, these interactions promote the cytoplasmic portion of TM4 shifting inward by ~2 Å (comparing the Cα of L3834.47 in FZD7–mGs with corresponding L3624.47 in SMO–Gi complex structures) (Supplementary information, Fig. S7f, black arrow).
A conserved molecular switch between TM6 and TM7 was previously identified for all class F GPCRs, maintaining the receptor in an inactive conformation (observed as a hydrogen-bonding distance between R6.32 and the backbone of W7.55) in all inactive class F receptor structures.7 The polar interactions between R6.32 and W7.55 are broken in active SMO–Gi and the FZD7–mGs complexes, resulting in a 6.4 Å distance between R4706.32 and W5477.55 in the FZD7–mGs complex (Fig. 1f, h).7
To explore the conformational dynamics around the open and active FZD7 structure, we performed molecular dynamics (MD) simulations of FZD7 in complex with mGs3935 (Supplementary information, Fig. S8). Monomeric mGs facilitated MD simulations due to its small size while minimizing the effect on receptor dynamics. These MD simulations allowed us to monitor general receptor integrity and the status of the molecular switch by assessing the angle of the kinked TM6 and the distance between R4706.32 and W5477.55. The overall hallmark of FZD7 activation — the kink in TM6 — is maintained over the time course of the simulation (measured as an angle between the backbone nitrogen atoms of V4856.47, P4816.43 and E4626.24). P6.43 is fully conserved among the FZD paralogues, but not in SMO (F6.43) (Supplementary information, Fig. S9). Analogous to P6.50 and P6.47 in class A and B receptors, respectively (Supplementary information, Fig. S10), P4816.43 is likely to contribute to the observed outward movement of the lower part of TM614 (Fig. 1f, h). In the MD trajectories, the conformational changes of TM6 are manifested by the disruption of the molecular switch and a rearrangement of an extended aromatic network stabilizing the active receptor conformation (Supplementary information, Fig. S8). R4706.32 and the backbone oxygen atom of W5477.55 remain at over 8 Å distance throughout the simulation, rendering hydrogen bonding impossible between these two residues. Instead, R4706.32 is frequently bound with the carboxyl terminus of LH5.26 of mGαs. Interestingly, R4706.32 remains within hydrogen-bonding distance to the carboxyl terminus of LH5.26 more often than K4666.28, indicating that these positively charged residues lock the carboxyl tail between them (Supplementary information, Fig. S8d). This could contribute to the observed non-helical conformation of the tail of the α5-helix.
To gather functional evidence for the FZD7–mGs interface and its role in maintaining the constitutive activity of FZD7 towards Gs, we employed a mutagenesis-based approach in combination with assessment of Gαs translocation and cAMP production as functional readouts of Gs-dependent signaling. We focused on D457 (in ICL3) and K4666.28, which interact with the α5-helix of mGαs. Mutating either D457 or K4666.28 to alanine alone did not affect the constitutive activity of FZD7 on Gs translocation or cAMP production (Fig. 1j, k; Supplementary information, Figs. S11, S12). However, the double mutant D457A/K4666.28A abrogated FZD7 constitutive activity towards Gs, suggesting that these mutations collectively interfere with G protein coupling. In contrast, the double mutant did not affect the ability of FZD7 to mediate WNT-induced activation of the WNT/β-catenin pathway as assessed by the TOPFlash reporter assay (Supplementary information, Fig. S13), underlining the concept of conformational selection for DVL-dependent signaling over G protein coupling as has been suggested previously.7,15
Although the CRD could not be resolved in the present structure, we observed that removal of the CRD (ΔCRD-FZD7) resulted in the inability to reconstitute the receptor–mGs complex in vitro to the same extent as that of full-length FZD7 (Supplementary information, Fig. S14). Thus, we surmised that the CRD is required for FZD7–mGs complex stability and that removal of the CRD could decrease constitutive activity. Therefore, we assessed the ability of the ΔCRD-FZD7 construct to functionally couple to Gs by assessing Gαs translocation and cAMP production (Fig. 1j, k). Removal of the CRD blunted the constitutive activity towards Gs signaling as evidenced by the lack of Gαs translocation and cAMP production. These data underline the requirement for the CRD to maintain constitutive activity of FZD7 towards heterotrimeric G proteins through intramolecular allostery.
In conclusion, we report the cryo-EM structure of FZD7–mGs demonstrating how constitutive activity feeds into downstream signaling via heterotrimeric G proteins. With respect to the overall diversity among GPCRs, FZD7 has evolved a unique way to maintain certain homologous movements consistent with class A and B GPCR activation, while adapting its class-specific architecture to mediate G protein activation. While the classical hallmarks of G protein engagement are present in our structure, several differences can be found at the interface between the receptor and the G protein suggesting that FZDs harbor their own selectivity determinants for heterotrimeric G proteins. In short, the present structure of constitutively active FZD7–mGs, alongside previously published inactive structures of FZD4 and FZD5, opens the door to more accurate modeling of other FZDs and a platform for in silico drug discovery, which will aid in the discovery of new treatments to help those afflicted with diseases of WNT-FZD signaling.
The cryo-EM 3D map of the FZD7–mGs–Nb35 complex has been deposited in EMDB database with accession code EMD-31340; the coordinates have been deposited in PDB database with accession code 7EVW. The MD simulation data is available at www.gpcrmd.org with ID 245.
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This work was supported by the National Key R&D Program of China (2018YFA0507000 to F.X.), and the National Natural Science Foundation of China (32071194 to F.X.). Work at Karolinska Institutet was supported by Karolinska Institutet, the Swedish Research Council (2017-04676; 2019-01190), the Swedish Cancer Society (CAN2017/561, 20 1102 PjF, 20 0264P), the Novo Nordisk Foundation (NNF17OC0026940; NNF20OC0063168), The Swedish Society of Medical Research (SSMF; P19-0055), the Lars Hierta Memorial Foundation (FO2019-0086, FO2020-0304), The Alex and Eva Wallström Foundation for Scientific Research and Education (2020-00228), and the German Research Foundation (DFG, 427840891; KO 5463/1-1). S.C.W. is supported by a fellowship from the Swedish Society for Medical Research (P18-0098). Computational resources were provided by the Swedish National Infrastructure for Computing (SNIC 2020/5-500). M.B. is funded by the CIHR (FDN-148431) and holds a Canada Research Chair in Signal Transduction and Molecular Pharmacology. We thank Qiwen Tan, Lu Zhang, Junlin Liu, Na Chen, Qiaoyun Shi and Wei Xiao from iHuman Institute for protein cloning and expression support; Qianqian Sun, Yunhun Liu and Zhihui Zhang at the Bio-EM facility at ShanghaiTech University for technical support on data collection. We thank Vadim Cherezov from University of Southern California for advice on structure refinement.
M.B. is the president of the scientific advisory board for Domain Therapeutics. M.B. has filed patent applications related to some of the biosensors used in this work and the technology has been licensed to Domain Therapeutics.
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Xu, L., Chen, B., Schihada, H. et al. Cryo-EM structure of constitutively active human Frizzled 7 in complex with heterotrimeric Gs. Cell Res (2021). https://doi.org/10.1038/s41422-021-00525-6