Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Cryo-EM structure of constitutively active human Frizzled 7 in complex with heterotrimeric Gs

Dear Editor,

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. S3S5, Table S1).

Fig. 1: Structure of constitutively active FZD7 in complex with heterotrimeric mGs.

a Normalized BRET0 values of ΔFZD1–10 HEK293 cells transiently co-transfected with the Gs BRET sensor along with either negative control (mock), the β2-adrenoceptor (β2AR) or FZD7. Data are represented as the means ± SEM of raw BRET0 that were obtained from simple linear regression of five independent experiments measured in quadruplicates shown in Supplementary information, Fig. S1A and normalized to the negative control. *P < 0.05; **P < 0.01 (one-way ANOVA followed by Sidak’s multiple comparison). b An annotated 2D class average of FZD7–mGs–Nb35 complex. c Overall density map and atomic model of FZD7–mGs–Nb35 complex (CRD was omitted due to linker flexibility). FZD7, blue; mGαs, orange; Gβ, green; Gγ, yellow; Nb35, gray. d Insertion of the α5-helix (mGαs, orange) into FZD7 helical bundle represented as surface (ICL1, blue; ICL2, pink; ICL3, yellow; TM7/H8, green). e Schematics of interactions between FZD7 and α5-helix. Hydrogen bonds are shown as red dashed lines. The red circle represents the hydrophobic interaction network. Yellow shades indicate residues that reside in TM5/6/ICL3; pink, TM3/4/ICL2; blue, TM1/2/ICL1; green, TM7/H8. f Superposition of FZD7 (blue) and FZD4 (yellow) structures, viewed from the intracellular side (bottom view). g Superposition of the active FZD7 structure (blue) with the inactive FZD4 (PDB: 6BD4, yellow), inactive FZD5 (PDB: 6WW2, light pink), active SMO (PDB: 6OT0, gray) and inactive SMO (PDB: 5V57, green) structures. h Comparison of the cytoplasmic portion of TM6 (from K6.28 to P6.43) in FZD7, FZD4, active SMO and inactive SMO structures. i R6.32, F6.36, W7.55 network in FZD7, FZD4 and active SMO structures. Blue dashed lines indicate the distance of F6.36–W7.55 and F6.36–R6.32 in FZD7. Gray dashed lines indicate the distance of R6.32–W7.55 and W7.55–F6.36 in active SMO structure. j Normalized BRET0 values of ΔFZD1-10 HEK293 cells transiently co-transfected with rGFP-CAAX and Gαs-67-RlucII, along with either negative control (mock), wild-type FZD7, ΔCRD-FZD7 or the indicated FZD7 mutants. Data are represented as the means ± SEM of raw BRET0 that were obtained from simple linear regression of four independent experiments measured in quadruplicates shown in Supplementary information, Fig. S11b and normalized to the negative control. **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison). k Normalized FRET0 values of ΔFZD1–10 HEK293 cells transiently co-transfected with the FRET-based cAMP biosensor along with either negative control (mock), wild-type FZD7, ΔCRD-FZD7 or the indicated FZD7 mutants. Data are represented as the means ± SEM of raw FRET0 that were obtained from simple linear regression of five independent experiments measured in quadruplicates shown in Supplementary information, Fig. S11c and normalized to the negative control. **P < 0.01; ****P < 0.0001 (one-way ANOVA followed by Sidak’s multiple comparison).

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.

Data availability

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 with ID 245.


  1. 1.

    Schulte, G. & Wright, SC. Trends Pharmacol. Sci. 39, 828–42 (2018).

    CAS  Article  Google Scholar 

  2. 2.

    Taipale, J. & Beachy, PA. Nature 411, 349–54 (2001).

    CAS  Article  Google Scholar 

  3. 3.

    Kozielewicz, P., Turku, A. & Schulte, G. Mol. Pharmacol. 97, 62–71 (2020).

    CAS  Article  Google Scholar 

  4. 4.

    Phesse, T., Flanagan, D. & Vincan, E. Cancers 8, (2016).

  5. 5.

    Nehmé, R., Carpenter, B., Singhal, A., Strege, A., Edwards, PC. & White, CF. PLoS ONE 12, e0175642 (2017).

    Article  Google Scholar 

  6. 6.

    von Maltzahn, J., Bentzinger, CF. & Rudnicki, MA. Nat. Cell. Biol. 14, 186–91 (2012).

    Article  Google Scholar 

  7. 7.

    Wright, SC., Kozielewicz, P., Kowalski-Jahn, M., Petersen, J., Bowin, CF. & Slodkowicz, G. et al. Nat. Commun. 10, 667 (2019).

    CAS  Article  Google Scholar 

  8. 8.

    Schihada, H., Shekhani, R. & Schulte, G. bioRxiv (2021).

  9. 9.

    Rasmussen, SG. et al. Nature 477, 549–55 (2011).

    CAS  Article  Google Scholar 

  10. 10.

    Kozielewicz, P. et al. Nat. Commun. 11, 414 (2020).

    CAS  Article  Google Scholar 

  11. 11.

    Yang, S. et al. Nature 560, 666–70 (2018).

    CAS  Article  Google Scholar 

  12. 12.

    Valnohova, J., Kowalski-Jahn, M., Sunahara, RK. & Schulte, G. J. Biol. Chem. 293, 17875–87 (2018).

    CAS  Article  Google Scholar 

  13. 13.

    Qi, X. et al. Nature 571, 279–83 (2019).

    CAS  Article  Google Scholar 

  14. 14.

    Turku, A., Schihada, H., Kozielewicz, P., Bowin, CF. & Schulte, G. Nat. Commun. (2021).

  15. 15.

    Bowin, CF., Inoue, A. & Schulte, G. J. Biol. Chem. 294, 11677–84 (2019).

    CAS  Article  Google Scholar 

Download references


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.

Author information




L.X. performed cloning, protein purification, cryo-EM sample preparation, data collection and structure analysis; B.C. performed cryo-EM data processing, model building and refinement; Y.W. assisted with the structure analysis and some calculations; G.W.H. was responsible for structure quality control; X.Z. and C.L. characterized the protein expression at early phase of the project; H.S. and S.C.W. performed functional biosensor experiments; M.K.J. and P.K. performed FZD7 construct mutagenesis for functional analysis; C.F.B. validated FZD7 surface expression; A.T. performed the MD simulation and analysis and contributed to model interpretation and visualization. F.X. conceived the project. F.X. and G.S. designed, coordinated and supervised the experiments. L.X., B.C., S.C.W., H.S., M.B., G.S. and F.X. wrote the manuscript.

Corresponding authors

Correspondence to Gunnar Schulte or Fei Xu.

Ethics declarations

Competing interests

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.

Supplementary information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation


Quick links