Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling

Naotaka Tsutsumi; Somnath Mukherjee; Deepa Waghray; Claudia Y Janda; Kevin M Jude; Yi Miao; John S Burg; Nanda Gowtham Aduri; Anthony A Kossiakoff; Cornelius Gati; K Christopher Garcia

doi:10.7554/eLife.58464

. 2020 Aug 7;9:e58464. doi: 10.7554/eLife.58464

Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling

Naotaka Tsutsumi ^1,^2,³, Somnath Mukherjee ⁴, Deepa Waghray ¹, Claudia Y Janda ^1,^2,⁵, Kevin M Jude ^1,^2,³, Yi Miao ^1,², John S Burg ^1,², Nanda Gowtham Aduri ^2,⁶, Anthony A Kossiakoff ⁴, Cornelius Gati ^2,^6,^✉, K Christopher Garcia ^1,^2,^3,^✉

Editors: Yi Arial Zeng⁷, Olga Boudker⁸

PMCID: PMC7442489 PMID: 32762848

Abstract

Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/β-catenin signaling in response to Wnt3a versus a ‘surrogate agonist’ that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/β-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine.

Research organism: Human

Introduction

Frizzled (Fzd) receptors recognize lipidated and glycosylated growth factors, called Wnts, that play crucial roles in stem cell biology, embryonic development and adult tissue regeneration (Nusse and Clevers, 2017). Although Fzd receptors are part of the class F G protein-coupled receptor (GPCR) family and involved in G protein signaling (Wright et al., 2018), they primarily activate the canonical β-catenin pathway (Carmon and Loose, 2008; He et al., 1997). Binding of Wnt to Fzd induces formation of a ternary signaling complex with the co-receptor LRP5/6, recruiting the intracellular complex comprised of Dishevelled (Dvl), Axin, APC, CK1 and GSK3. This event silences the destruction complex, leading to accumulation of transcriptional co-activator β-catenin and the induction of Wnt target genes. Because aberrant activation of Wnt/β-catenin signaling can lead to tumorigenesis, inhibition of Wnt signaling is a prime target as a cancer therapy (Anastas and Moon, 2013). Furthermore, Wnt’s essential function in orchestrating stem cell proliferation and differentiation have also prompted interest in its use to promote tissue regeneration upon injury (Clevers et al., 2014).

Unlike class A GPCRs, where ligand-induced allostery is well established, the structural and mechanistic basis of Wnt signaling through Fzd remains enigmatic. Wnt signaling requires both a seven transmembrane (TM) GPCR-like (Fzd) and a 1TM (LRP5/6) receptor. On one hand, the clear similarity of Fzd to GPCRs suggests that ligand-induced conformational change could be at play (Wright et al., 2018). On the other hand, canonical Wnt/β-catenin signaling requires Fzd cross-linking with LRP5/6 by both Wnts and Norrin, implying a heterodimerization mechanism analogous to single-pass TM receptors such as cytokine receptors (Janda et al., 2017). To what extent Wnt-induced conformational changes versus simple Fzd/LRP6 heterodimerization play a deterministic role in signaling is not completely clear. Moreover, although structural information on the Fzd cysteine-rich domain (CRD) and its recognition mechanism with both Wnts and Norrin (Chang et al., 2015; Hirai et al., 2019; Janda et al., 2012) is available, a structural understanding of the Fzd-7TM regions has lagged behind (Zhang et al., 2018).

The class F GPCRs include Smoothened (Smo) and ten Fzd family members which are classified into five subgroups based on homology and Wnt specificity: Fzd1/2/7, Fzd3/6, Fzd4, Fzd5/8 and Fzd9/10 (MacDonald and He, 2012). The overall architecture of Fzd receptors consists of an N-terminal CRD, or ligand-binding domain, that serves as the Wnt binding module, and a seven-transmembrane domain (7TM) that is connected to the CRD by a long hinge region (Zhang et al., 2018; Figure 1—figure supplement 1). The hinge region can be further subdivided into a flexible segment from the CRD to the first conserved cysteine residue, and the ordered segment from the cysteine to TM1, with the total lengths varying from 50 to 115 amino acid residues. The hFzd4-7TM structure, lacking the CRD, has been recently reported along with structure-based mutagenesis data and molecular dynamics simulations (Yang et al., 2018), but it contains thermostabilized mutations that make it difficult to ascertain the relevance of the TM conformations in comparison to Smo and class A GPCRs.

In order to deduce insights into the structural basis of Fzd activation, we determined a fiducial-assisted cryo-EM structure at 3.7 Å resolution of hFzd5, lacking any thermostabilizing mutations. We also analyzed Wnt/Fzd complex particles that revealed a flexibly connected Wnt/CRD module that does not allow us to resolve a high-resolution structure of a stable binary Wnt/Fzd complex. The Fzd-TM structure guided the design of a series of mutations to explore the role of transmembrane coupling in signaling by both Wnt and a surrogate agonist. The identical structure-activity relationships shown by the natural versus the synthetic bi-specific agonist strongly suggest that Wnt/β-catenin signaling through Fzd is largely dependent on cross-linking with LRP6, rather than class-A GPCR-like conformational changes. Thus, the canonical Wnt/β-catenin signaling mechanism is more reminiscent of ligand-induced heterodimerization of single-pass receptors, such as cytokine and receptor Tyrosine kinases (RTK). Understanding this signaling mechanism is key to utilizing the canonical Wnt/β-catenin pathway in regenerative medicine.

Results and discussion

Structure determination and analysis

The initial attempts at reconstructing the 65 kDa hFzd5 molecule were hampered due to the particle shape being dominated by the spherical features of the detergent micelle, an issue that complicates structure determination of small membrane proteins by cryo-EM. To facilitate unambiguous alignment of the particles, we sought to introduce features into the particle that would both increase its size and provide an asymmetric shape that would enable facile alignment for 3D reconstruction of hFzd5. We introduced the BRIL domain (cytochrome b562 RIL) fused in place of intracellular loop 3 of hFzd5. In order to optimize a rigid connection between hFzd5 and BRIL, we grafted two short linkers, each comprising five amino acid residues from A_2A adenosine receptor-TMs between TM5/6 of Fzd5 and BRIL (See Methods), as used for the first full-length Smo structure (Byrne et al., 2016). We employed an anti-BRIL Fab, BAG2 (Mukherjee et al., 2020; Figure 1a) with an anti-Fab nanobody (Nb) (Ereño-Orbea et al., 2018) to complex with the BRIL fusion hFzd5 (hFzd5_ICL3BRIL). The BRIL/Fab/Nb module was designed as a ‘fiducial’ marker for particle image alignment (Mukherjee et al., 2020). We purified hFzd5_ICL3BRIL from 293S GnTI^- cells in a glycol-diosgenin/cholesterol hemisuccinate (GDN/CHS) micelle and incubated it with excess Fab and Nb before the size-exclusion chromatography step (Figure 1—figure supplement 2a). The peak fraction of the complex was concentrated to 8 mg/ml with digitonin as an additive for single-particle cryo-EM analysis (Figure 1—figure supplements 2b,c and 3).

Figure 1. — (a) Cartoon representation of the strategy for hFzd5 particle decoration by anti-BRIL Fab and anti-Fab Nb. These chaperones double hFzd5 molecular weight and render it asymmetric in detergent micelle, thus working as a fiducial maker for image alignment. (b) A selected 2D class average showing the side view of monomeric hFzd5_ICL3BRIL/Fab/Nb. (c) Overall EM volume around the structural model of hFzd5 (cyan), BRIL and the linker between BRIL and Fzd5 (light blue), Fab heavy chain (pink), Fab light chain (light pink) and Nb (wine). (d) The size-exclusion chromatography profile, SDS-PAGE of peak fractions, and (e) the selected 2D class average of XWnt8/hFzd5FL. The full SDS-PAGE and 2D classes are shown in Figure 1—figure supplement 5. Models of XWnt8/mouse Fzd8CRD (mFzd8CRD) (PDB ID: 4F0A) and hFzd5 are overlaid on the blobs to show the relative size of densities.

Figure 1—figure supplement 1. — (a) Cartoon representation of the strategy for hFzd5 particle decoration by anti-BRIL Fab and anti-Fab Nb. These chaperones double hFzd5 molecular weight and render it asymmetric in detergent micelle, thus working as a fiducial maker for image alignment. (b) A selected 2D class average showing the side view of monomeric hFzd5_ICL3BRIL/Fab/Nb. (c) Overall EM volume around the structural model of hFzd5 (cyan), BRIL and the linker between BRIL and Fzd5 (light blue), Fab heavy chain (pink), Fab light chain (light pink) and Nb (wine). (d) The size-exclusion chromatography profile, SDS-PAGE of peak fractions, and (e) the selected 2D class average of XWnt8/hFzd5FL. The full SDS-PAGE and 2D classes are shown in Figure 1—figure supplement 5. Models of XWnt8/mouse Fzd8CRD (mFzd8CRD) (PDB ID: 4F0A) and hFzd5 are overlaid on the blobs to show the relative size of densities.

The vast majority of 2D class averages of the ‘fiducial-assisted’ Fzd5 were monomeric particles with the Fab/Nb module clearly discernable, but with sparse density for the Fzd-CRD (Figure 1b and Figure 1—figure supplement 2c). It has been suggested that Fzd receptors are capable of forming homodimers in both Wnt-dependent and -independent manners (Carron et al., 2003; Hirai et al., 2019; Nile et al., 2017). We observed a small population (~5% of the monodisperse particles) of parallel hFzd5 dimers in the 2D class averages (Figure 1—figure supplement 2c, marked with red boxes), which places the ICL3BRIL/Fab/Nb units on opposite sides of the dimeric receptor. The relative position of two Fabs indicates the TM4s are likely at the dimer interface given the expected GPCR fold, consistent with the crystal structure of hFzd4TM (Yang et al., 2018). Since Fzd appears to be monomeric on the surface of live cells, and hetero-trimerization of Wnt/Fzd with LRP6 is sufficient for activation of the intracellular β-catenin signaling (Janda et al., 2017), the role of observed hFzd5 dimers in signaling remains unclear (Tao et al., 2019).

3D classification of selected monomeric particles indicated the fusion between the TM region and BRIL was not completely rigid (Figure 1—figure supplement 3a). Two particle classes with clear TM density and well-defined features of the Fab/Nb module showed slightly varied angles (approximate median angular variation of 2^o). We initially focused on the class with the highest resolution and most clearly resolved extracellular features from the final heterogeneous refinement step, yielding a 3D map with a nominal resolution of 4.6 Å at the Fourier shell correlation (FSC) criterion of 0.143. To overcome the residual flexibility between hFzd5 and BRIL, local non-uniform refinement was performed in cryoSPARC (Punjani et al., 2017) with a fulcrum on the hFzd5-BRIL linker. The final 3D map was created by combining the two different classes, resulting in a 3.7 Å map of monomeric hFzd5_ICL3BRIL/Fab/Nb with substantially better TM features allowing model building (Figure 1c, Table 1 and Figure 1—figure supplements 3 and 4).

Table 1. Cryo-EM data collection and refinement statistics.

	hFzd5_ICL3BRIL/anti-BRIL Fab/anti-Fab Nb (EMD-21927) (PDB ID: 6WW2)
Data collection and processing
Magnification	81,000x
Voltage (kV)	300
Electron exposure (e/Å²)	50
Defocus range (μm)	−0.8 to −2.0
Pixel size (Å)	1.078
Symmetry imposed	C1
Initial particle images (no.)	6,483,398
Final particle images (no.)	369,704
Map resolution (Å) FSC threshold	3.70 0.143
Map resolution range (Å)	3.2 to 5.9
Refinement
Initial model used (PDB code)	5L7D, 6BD4, 6ANI, 6CBV
Model resolution (Å) FSC threshold	3.7/4.1 0.143/0.5
Map sharpening B factor (Å²)	−124.4
Model composition Non-hydrogen atoms Protein residues	7,594 982
B factors (Å²) Protein	89.59
R.m.s. deviations Bond lengths (Å) Bond angles (°)	0.010 1.083
Validation MolProbity score Clashscore Poor rotamers (%)	1.87 5.73 0
Ramachandran plot Favored (%) Allowed (%) Disallowed (%)	89.59 10.31 0.10

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The EM volume is defined for the extracellular loops (ECL) 1–2 and the hinge domain (C192-F227). However, the CRD, the CRD-proximal flexible hinge and a part of ECL3 (A27-Y150, N151-K191 and P487-P498, respectively) are either missing or ambiguous in the 3D reconstruction, suggesting intrinsic flexibility between the 7TM and the ligand binding-module. Given the flexibility between the CRD and 7TM regions and their lack of intimate structural coupling, it seems unlikely that Wnt binding to CRD domain could be propagated through a conformational change to the 7TM bundle to modulate signaling in a manner analogous to authentic GPCRs. Indeed, the hinge of Fzd8, a Fzd5/8 subfamily member, is also quite extended and is ~40 amino acid residues longer than the one of Fzd5, but they share similar Wnt specificity and signaling properties (Voloshanenko et al., 2017).

We also imaged Xenopus Wnt8 (XWnt8) bound to full-length hFzd5 (hFzd5FL) without the BRIL fusion, the Fab, or the Nb (Figure 1d,e and Figure 1—figure supplement 5). After successfully purifying a stable complex, 2D class averages revealed two rather blurred densities that match the size of Wnt/Fz in detergent micelle. However, the densities for both regions are limited to low resolution due to extreme segmental variation and flexibility, and therefore do not allow us to resolve a complete structure of the complex. It is likely that LRP6, missing in our complex, could stabilize the Wnt/CRD module. Nevertheless, an approximate placement of hFzd5FL and XWnt8 in the 2D class averages reveals Wnt projecting straight up from the Fzd-TM-micelle, with a narrow stricture of missing density for the linker region. These 2D classes represent an average relative position of the two molecules that exist in a spectrum of angular variations. This allowed us to visualize both Wnt and Fzd densities, but probably represent only a part of many alternative Wnt/Fzd-CRD positions in the total population of particles. Attempts of 3D classification and reconstruction were not successful due to their severe segmental heterogeneity. Despite the limitations of the Wnt/Fzd data, the particles further support the conclusion that the Wnt/Fzd-CRD module is not in intimate contact with the TM-regions of Fzd and is therefore unlikely to participate in a tightly structurally coupled allosteric relay upon Wnt binding.

Although Smo has one of the shortest hinges among human Class F GPCRs, the cryo-EM structure of the Smo/G protein complex showed the EM map of the CRD was not well-defined (Qi et al., 2019). Nevertheless, the crystal structures of full-length Smo showed that the CRD could ‘sit’ on the ordered extracellular loops (Byrne et al., 2016) suggesting Smo-CRD is less flexible than Fzd-CRD. The freedom of the Fzd-CRD should enable Fzd to ‘search’ for ligands encountered in the membrane from the side of the receptor, not just from the top.

Structural comparison between hFzd5, hFzd4 and hSmo

Human Fzd5 belongs to a distinct subfamily from hFzd4, but they share high similarity with the 7TM GPCR fold and have ~62% sequence identity in TMs (excluding the extracellular TM6-ECL3 residues 6.58–6.77, generic numbering for class F GPCRs Isberg et al., 2015). In the Fzd5 structure, the ECL2 forms a lid comprised of β-sheets and loops (Figure 2a,b). The hinge region forms an ordered structure that, along with ECLs, occludes the orthosteric ligand binding pocket defined for Smo and class A GPCRs. In contrast to the lipid GPCRs (EP3, EP4, and TP) that possess complete ECL2 lids with a gap between TMs (Audet et al., 2019; Fan et al., 2019; Morimoto et al., 2019; Toyoda et al., 2019), neither the Fzd5 nor Fzd4 structure shows a large opening within the TMs, implying the receptors’ pocket would likely occlude small molecules. There are pronounced differences in the structure of the extracellular region of Fzd5, compared to the ECL structures of Fzd4 and Smo (Figure 2c). The hinge and linker domain of Fzd5 are more extended than those of Fzd4 and Smo, which are the shortest among the Class F GPCRs. The hinge domains of Fzd5/8 contain five cysteine residues, in contrast to three cysteines for the other subclasses in the Fzd/Smo family. Three cysteine residues in the flexible hinge and the start of the hinge domain are unmodeled in the structure.

Figure 2. — (a) Top (left) and bottom (right) views, and (b) side views of hFzd5 colored by rainbow with blue on the N-terminus and red on the C-terminus of the structural model. (c) Comparison of the extracellular regions between hFzd5, hFzd4 (pink, PDB ID: 6BD4) and hSmo (yellow, PDB ID: 5L7D). Transparent surfaces are overlaid on the cartoon models and colored separately by the hinge domain (blue), ECL1 (green), ELC2 (wheat) and ELC3 (orange). XWnt8/mFzd8CRD structure (PDB ID: 4F0A) is displayed and connected to the hFzd5 structure by dashed line, to show the size of the LRP6 binding module relative to 7TM. The α-helices at TM6-ECL3 are indicated with gray dashed lines. (d) Top views of hFzd5, hFzd4, and hSmo around the Smo-7TM ligand binding site with potential gatekeeping residues. The cavity volume calculated using the CavityPlus server (Xu et al., 2018) are displayed as transparent surface representations, with a focus on the constrictions made by bulky resides of Fzd at the upper-core site. The antagonist vismodegib (from PDB ID: 5L7I) is overlaid on the hSmo structure in its inactive and *apo* state (PDB ID: 5L7D).

Figure 2—figure supplement 1. — (a) Top (left) and bottom (right) views, and (b) side views of hFzd5 colored by rainbow with blue on the N-terminus and red on the C-terminus of the structural model. (c) Comparison of the extracellular regions between hFzd5, hFzd4 (pink, PDB ID: 6BD4) and hSmo (yellow, PDB ID: 5L7D). Transparent surfaces are overlaid on the cartoon models and colored separately by the hinge domain (blue), ECL1 (green), ELC2 (wheat) and ELC3 (orange). XWnt8/mFzd8CRD structure (PDB ID: 4F0A) is displayed and connected to the hFzd5 structure by dashed line, to show the size of the LRP6 binding module relative to 7TM. The α-helices at TM6-ECL3 are indicated with gray dashed lines. (d) Top views of hFzd5, hFzd4, and hSmo around the Smo-7TM ligand binding site with potential gatekeeping residues. The cavity volume calculated using the CavityPlus server (Xu et al., 2018) are displayed as transparent surface representations, with a focus on the constrictions made by bulky resides of Fzd at the upper-core site. The antagonist vismodegib (from PDB ID: 5L7I) is overlaid on the hSmo structure in its inactive and *apo* state (PDB ID: 5L7D).

Compared with the extremely short ECL3 of Fzd4 comprising a small α-helix sitting parallel to the membrane, Fzd5 has the longer ECL3, annotated as a part of TM6 by sequence similarity with Smo. However, compared to Smo, Fzd5-ECL3 forms a shorter α-helix with an extended flexible loop that is not clearly defined in the EM map (Figure 2c and Figure 1—figure supplement 4c), which might favor ligand-induced allostery in Smo that is not seen in Fzd. For example, it has been proposed that during Smo signaling, the long TM6-ECL3 α-helix interacts with the CRD to sense ligand-induced conformational rearrangement by the CRD. This could facilitate allosteric communication between the CRD, TM6 and TM7 for receptor activation (Deshpande et al., 2019; Huang et al., 2018). Fzd5/8-ECL3 has a CXC motif that forms either a rare intra-loop disulfide bond or two disulfide bonds with the unique orphan cysteines in the flexible hinge (Figure 1—figure supplement 1). Smo and most Fzds have two cysteines dispersed in their ECL3s, and Smo shows an intra-ECL3 disulfide bond in the structure. In contrast, there is no ECL3 cysteine for Fzd4. The differences in cysteine distribution in the hinge region and ECL3 between Fzd4 and Fzd5 might explain the cell signaling data from a chimeric Fzd4, where the whole hinge region was replaced by the Fzd5 hinge (Bang et al., 2018). Although cell-surface expression was confirmed, the chimeric receptor was completely inactive to Norrin stimulation, and it was concluded that the loss of the Norrin-Fzd4 hinge interaction disrupted signaling. However, a more plausible explanation may be that the disruption of the disulfide network impaired the structural integrity of the chimeric receptor. Consistent with this explanation, several Fzd4 mutations of the hinge cysteines (C181R, C204Y, and C204R) render Fzd4 inactive in patients with familial exudative vitreoretinopathy (Milhem et al., 2014).

In Fzd-7TM, the ‘orthosteric’ pocket for small-molecules (eg. vismodegib, Anta XV, TC114, LY2940680 and SAG1.3 for Smo-7TM) has a constriction that restricts ligand access (Figure 2d). In the putative ‘druggable’ pocket, Fzd5 has a bulky ionic residue K506^7.41 (superscript denotes generic numbering for class F GPCRs residue). This residue is conserved in ten Frizzled members, potentially restricting the small molecule access together with the neighboring bulky sidechain of Y507^7.42. The approximate minimum diameters of the pockets are 4.9 Å for Fzd5 and 4.2 Å for Fzd4, whereas Smo has a 5.8 Å diameter stricture near the entrance of the cavity. Alanine substitution of either K506^7.41 or Y507^7.42 severely impaired cell surface protein expression of full-length hFzd5, implying the importance of these residues for protein stability likely through inter-helical packing (Figure 2—figure supplement 1). While the small pockets evident in the Fzd-7TM structures might help to explain the difficulty of developing small molecule modulators for Wnt/β-catenin signaling, a recent report showed the Smo agonist SAG1.3 acted as a partial agonist for Fzd6-Gi signaling (Kozielewicz et al., 2020). Thus, small molecule signal modulation of Fzd remains an area of active investigation.

We also compared the cytoplasmic regions of the 7TM bundles of Fzd5, Fzd4 and Smo, which are the regions that interact with signaling adaptor proteins. TM6 plays an important regulatory role in the activation of class A GPCRs, and Smoothened in the context of G protein signaling, and is found in a similar position in both Fzd4 and Fzd5. However, the cytoplasmic end of Fzd5-TM5 is in an inward position compared to Fzd4-TM5 and aligns well with the Smo-TM5 in its inactive state (Figure 3a). In this region, Fzd5 and Smo have a phenylalanine residue in TM5 or TM6 that interacts with W^3.50. Additionally, there is a small aliphatic residue on the counterpart TM6 or TM5, respectively that apparently stabilizes the ‘inward’ conformation. Fzd4 has L410^5.66 and V442^6.38 at the corresponding positions that might result in looser packing between TM5-TM6.

Figure 3. — (**a, b**) Structural comparison of hFzd5, hFzd4 and hSmo (a) at the cytoplasmic end of TM5, and (b) the K/R^6.32-W^7.55 ionic lock with the aromatic network around H8. (c) Bottom views of hFzd5 and hFzd4 showing structural rearrangement of ICL1 and H8 important for Dvl binding. Each molecule is colored by cyan (hFzd5), pink (hFzd4, PDB ID: 6BD4) or yellow (Smo, PDB ID: 5L7D).

An important question that could be related to receptor activation pertains to conformational changes of the Fzd-TM. For Fzd4, small distortions of the conformations of TM4 and TM7, including two kinks in TM7, were noted after molecular dynamics simulations of the crystal structure, leading to conjecture that TM4 and TM7 transitioned between two conformations to modulate signaling (Yang et al., 2018). TM4s and TM7s of Fzd4 and Fzd5 are closely structurally aligned, although Fzd5 W520^7.55 forms a more extensive π-cation interaction with R449^6.32 (W494^7.55 and K436^6.32 in Fzd4) (Figure 3a,b). The K/R^6.32-W^7.55 interaction, known as the ‘ionic lock’ is a crucial molecular switch for intracellular signaling in class F GPCRs, and it has been suggested that the Dvl- versus G protein-biased states exist with the K/R^6.32-W^7.55 interaction either on or off, respectively (Wright et al., 2019). R449A^6.32 in ‘canonical’ Fzd5 abolished Dvl recruitment and Wnt3a-induced β-catenin signaling, while R416A^6.32 in ‘non-canonical’ Fzd6 increased G protein signaling (Wright et al., 2019). Fzd5-H8, which has another Dvl-binding KTxxxW motif, undergoes a helical half-turn outward transition compared with Fzd4 (Figure 3c). The KTxxxW motif is known to interact with the PDZ domain and the DEP domain of Dvl (Gammons et al., 2016; Wong et al., 2003), where point mutations of the conserved K, T and W residues impair the canonical β-catenin signaling.

Probing the effect of structure-guided hFzd5 mutations on signaling

Does Wnt contain ‘intrinsic’ structural properties necessary to activate canonical signaling beyond simply acting as a Fzd/LRP6 cross-linker? Delineating between a Wnt-induced allosteric mechanism through Fzd, a la’ GPCR, and a simple Fzd/LRP heterodimerization mechanism remains challenging. Thus, we approached this experiment by comparing structure-activity relationships for canonical signaling induced by the natural ligand Wnt3a, versus a synthetic agonist that cross-links Fzd5 and LRP6 (Figure 4a,b; Janda et al., 2017). ‘Surrogate Wnt’ molecules serve as cross-linkers of Fzd and LRP6 receptors to form signaling heterodimers, but with distinct binding modes compared to Wnt, and therefore are devoid of any ‘intrinsic’ structural features of Wnt that might be contributing to an allosteric mechanism. We examined β-catenin/T cell factor (TCF) signaling downstream of hFzd5 upon stimulation with either hWnt3a or a surrogate Wnt using the Fzd1/2/4/5/7/8 knockout cell line (Voloshanenko et al., 2017; Figure 4 and Figure 2—figure supplement 1). We chose mutants from the Fzd5-TM structure that appear to mediate important inter-helical interactions that might be associated with allostery and have been previously noted in Fzd. K506A^7.41 and Y507A^7.42 mutation completely or severely impaired cell-surface Fzd expression, respectively, whereas other mutants showed comparable expression levels to the wild-type receptor (Figure 2—figure supplement 1). With wild-type Fzd5 expression, the cells signaled in response to Wnt3a and the surrogate Wnt in a concentration-dependent manner, confirming that the output signaling is Fzd5 specific (Figure 4a,b). We then tested a series of mutants in this assay system. Fzd5-Y507A^7.42 signaled as robustly as the wild-type receptor regardless of its low expression, indicating the expression levels are not limiting signaling strength. The ionic lock mutants Fzd5-R449A^6.32 and Fzd5-W520A^7.55 completely ablated or significantly reduced the signaling stimulated by both the agonists, in accordance with previous studies showing the importance of this interaction for signaling (Wright et al., 2019). W522A^7.55 and T252F^1.49/T534F^8.58 mutants did not alter signaling compared to wild-type Fzd5, while W238A^1.35 showed slightly lower signal compared to wild-type Fzd5 for both agonists. The V281Y^2.54 mutation, which is at the small molecule binding site in Smo, yielded a lower signal with Wnt3a stimulation, but a stronger signal for the surrogate Wnt than the wild type. In general, the response patterns were similar between Wnt3a and surrogate Wnt. In a previous mutational analysis of Fzd4 signaling by Wnt3a or Norrin, a generally similar signaling pattern was observed for both ligands (Yang et al., 2018).

Schematic presentation of the ternary complex formation, and hFzd5-mediated signaling upon stimulation by (a) Wnt3a and (b) surrogate Wnt agonist. Non-lipidated, water-soluble surrogate Wnt would form the complex in a different geometry from natural Wnt ligands. Each series was stimulated (a) by Wnt3a at the concentrations of 0, 1.6, 3.1, 6.3 or 12.5 nM, or (b) by surrogate Wnt at the concentrations of 0, 0.25, 1, 4 or 16 nM, displayed from left (no agonist) to right (the highest concentration) and indicated by black triangles. The bars and error bars represent means and standard deviations, respectively, of data points from three technical replicates shown as circles.

Figure 4—source data 1. Raw data points for the signaling experiments shown as circles in Figure 4.

elife-58464-fig4-data1.xlsx^{(13KB, xlsx)}

Collectively these results strongly imply that Wnt does not initiate conformational changes in Fzd that are intrinsic to Wnt, and that Wnt principally acts as a cross-linker. This is further supported by the long and flexible linker between the Fzd-TM and CRD, which does not allow intimate structural coupling between ligand binding and conformational changes in the TM. This stands in contrast to Smo, which has a more rigid linker, allowing allosteric signal transduction by sensing the stimulus on CRD. It is possible that G protein signaling is more reliant on the conformational change of Fzds. Also, it is still unclear if the proximity of LRP6 to Fzd induces conformational changes in Fzd as a result of heterodimerization, but if so, this would be driven by structural properties intrinsic to the receptors, not Wnt. The canonical Wnt system, comprising both a 7TM GPCR-like receptor (Fzd) and a single-pass 1TM receptor (LRP5/6), has presented a mechanistic conundrum in that GPCRs signal through ligand-induced allostery, whereas single-pass TM receptors generally signal via ligand-induced dimerization, exemplified by RTK and JAK/STAT cytokine receptors (Wilmes et al., 2020). In this case, the preponderance of structural and mechanistic data point to a unique example of a heterodimerization-driven mechanism by a GPCR-like receptor Fzd and a Type-I receptor LRP5/6. This feature opens up the possibility of ‘tuning’ Wnt signaling through the use of alternative agonist molecules, such as antibodies and other dimeric scaffolds (Tao et al., 2019), to obtain a more granular understanding of Wnt biology and for developing regenerative therapeutics.

Materials and methods

Key resources table.

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Cell line (Spodoptera frugiperda)	Sf9	ATCC	CTL-1711 (RRID:CVCL_0549)	Insect cells used for baculovirus production
Cell line (Homo sapiens)	HEK293S GnTI^-	Gift from Prof. H Gobind Khorana (PMID:12370423)		Mammalian cells used for baculovirus expression of hFzd5FL and hFzd5_ICL3BRIL
Cell line (Homo sapiens)	HEK293T with Fzd1/2/4/5/7/8 KO	Gift from Prof. Michael Boutros (PMID:28733458)		For signaling assay
Cell line (Mus musculus)	Mouse monoclonal M1 hybridoma	Gift from Prof. Brian K Kobilka (PMID:17962520)		To purify anti-FLAG M1 (mouse IgG2a) antibody to prepare FLAG affinity column (15 mg/ml resin). This was used for purification of hFzd5FL and hFzd5_ICL3BRIL.
Strain, strain background (Escherichia coli)	BL21(DE3)	New England Biolabs	C2527I	E. coli strain for expression of the nanobody
Strain, strain background (Escherichia coli)	BL21 (gold)	Agilent Technologies	230130	E. coli strain for expression of the Fabs
Transfected construct (Homo sapiens)	pRK5 hFzd5 wild-type	Yu et al., 2012 (PMID:23095888)		For signaling assay
Transfected construct (Homo sapiens)	pRK5 hFzd5 mutants	this study		For signaling assay
Recombinant DNA reagent	7xTCF-Ffluc	Addgene	24308	For signaling assay
Recombinant DNA reagent	BestBac Linearized Baculovirus DNA 2.0	Expression Systems	91–002	For baculovirus production
Peptide, recombinant protein	Wnt3a	R and D Systems	5036-WN	For signaling assay
Peptide, recombinant protein	Surrogate Wnt	Miao et al. Cell Stem Cell 2020		For signaling assay
Peptide, recombinant protein	DRPB_Fz8	Dang et al., 2019 (PMID:31086346)		For signaling assay
Commercial assay or kit	Luciferase assay kit	Promega	E152A	For signaling assay
Chemical compound, drug	n-Dodecyl-β-D-Maltoside (DDM)	Anatrace	D310S	Membrane protein purification
Chemical compound, drug	Lauryl Maltose Neopentyl Glycol (LMNG)	Anatrace	NG310	Membrane protein purification
Chemical compound, drug	Glyco-Diosgenin (GDN)	Anatrace	GDN101	Membrane protein purification
Chemical compound, drug	Cholesterol Hemisuccinate tris Salt (CHS)	Anatrace	CH210	Membrane protein purification
Chemical compound, drug	Digitonin	EMD Millipore	300410	Cryo-EM specimen freezing additive
Software, algorithm	DigitalMicrograph	Gatan		Microscope alignment and cryo-EM data collection
Software, algorithm	serialEM	https://bio3d.colorado.edu/SerialEM/ (PMID:16182563)	version 3.6	Cryo-EM data collection
Software, algorithm	MotionCor2	https://emcore.ucsf.edu/ucsf-motioncor2/ (PMID:28250466)		Motion correction of cryo-EM movies
Software, algorithm	cryoSPARC	https://cryosparc.com/ (PMID:28165473)	version 2.12.14	Cryo-EM data processing
Software, algorithm	Coot	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ (PMID:20383002)		Structure model building
Software, algorithm	Isolde	https://isolde.cimr.cam.ac.uk/ (PMID:29872003)		Structure model building/refinement
Software, algorithm	Phenix suite	https://www.phenix-online.org/ (PMID:20124702)		Structure refinement
Software, algorithm	UCSF Chimera	https://www.cgl.ucsf.edu/chimera/ (PMID:15264254)		Initial homology model docking
Software, algorithm	UCSF ChimeraX	https://www.rbvi.ucsf.edu/chimerax/ (PMID:28710774)		Structural visualization/figure preparation/Isolde execution
Software, algorithm	PyMol	Schrödinger		Structural visualization/figure preparation
Software, algorithm	GraphPad Prism 7	GraphPad		analysis of signaling data
Other	CNBr-Activated Sepharose 4 Fast Flow	GE Healthcare	17098101	For preparation of anti-FLAG sepharose
Other	Lipofectamine 2000	Invitrogen	11668030	Transfection reagent for signaling assay

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Cell lines

Sf9 cells were obtained from ATCC (CTL-1711, RRID:CVCL_0549), HEK293S GnTI^- cells were provided by Prof. H Gobind Khorana, Fzd1/2/4/5/7/8 knockout HEK293T cells were provided by Prof. Michael Boutros and M1 hybridoma cells were provided by Prof. Brian K Kobilka.

Cell line authentication was guaranteed by the sources where the cells were obtained. For Sf9, ATCC does not indicate the authentication method. For HEK293S GnTI^- cells, clonal selection was performed by the provider based on their GnTI activity, and the authentication was regularly monitored by purified protein's sensitivity to Endo H enzyme. For Fzd1/2/4/5/7/8 knockout HEK293T cells, the original cell authentication was confirmed with NGS sequencing by the provider, and the authentication was regularly monitored by their response to Wnt3a as shown in this study (negative and positive controls). For M1 hybridoma, clonal selection was performed by the provider based on the antibody expression levels, and the authentication was monitored in the same way. Routine mycoplasma tests were not performed for each cell line. All cell lines were kept at low passages in order to maintain their health and identity.

Protein expression and purification for cryo-EM study

The expression construct for human Frizzled five was designed with insertion of cytochrome b562 RIL (BRIL) in the intracellular loop 3 (termed hFzd5_ICL3BRIL) with no additional thermostabilizing mutation. The chimeric protein was made using the inactive BRIL-fusion Smoothened with V329F^3.40 mutation in TM3 (PDB: ID 5L7D) as a reference, which yielded well-diffracting crystals in the report (Byrne et al., 2016). Wild-type hFzd5 (residue 27–428 and 441–546, UniProt ID: Q13467) was connected to BRIL using two short linkers derived from A_2A adenosine receptor (ARRQL between residue 428 and N-terminus of BRIL, and ARSTL between C-terminus of BRIL and residue 441) and cloned with an N-terminal HA signal peptide and a FLAG epitope into the mammalian baculovirus expression (BacMam) vector pVLAD6 (Dukkipati et al., 2008), that has a C-terminal 3C protease site followed by a protein C epitope and an octahistidine tag. While we did not have to test multiple constructs for cryo-EM screening, we noticed that the extended helical linker was suitable for the Fab marker, which would have otherwise led to a ‘collision’ between the Fab/Nb unit and the detergent micelle. We also note that hFzd5_ICL3BRIL had an overall 5-fold higher yield after expression and purification compared to hFzd5FL, indirectly suggesting its superior stability.

P0 virus was prepared in Sf9 by co-transfecting the expression vector and BestBac Linearized Baculovirus DNA (Expression Systems, CA, USA) using a standard method, and the virus was amplified from P0 to P2. hFzd5_ICL3BRIL was expressed in HEK293S GnTI^- cells with BacMam baculovirus transduction. 4% (v/v) P2 virus was added to cells at a density of 2 × 10⁶ cells per ml and culture flasks were shaken at 37 °C for 24 hr with 5% CO₂. After centrifugation, cells were washed with phosphate-buffered saline (PBS) supplemented with 5 mM EDTA and 1:1000 protease inhibitor cocktail (PIC, Sigma Aldrich, MO, USA), weigh and stored at −20°C. Approx. 30 grams of cell pellet was thawed and lysed with a Dounce homogenizer in a lysis buffer composed of 20 mM Tris-HCl pH 8.0, 5 mM EDTA, 2 mg/ml iodoacetamide, and 1:1000 PIC. The lysate was centrifuged at 48,000 x g for 1 hr and the membrane pellet was resuspended and nutated for 2 hr in 400 ml of a solubilization buffer consisting of 10 mM HEPES pH 7.2, 150 mM NaCl (HBS), 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Sol-Grade, Anatrace), 0.2% (w/v) cholesterol hemisuccinate (CHS, Anatrace), 10% (v/v) glycerol, 2 mg/ml iodoacetamide, and cOmplete PIC (Roche, Basel, Switzerland).

After centrifugation at 48,000 x g for 1 hr, 10 ml Ni-NTA resin (Qiagen, Hilden, Germany) was added to the supernatant, and the mixture was stirred at 4°C overnight. The resin was collected in a column, washed with HBS with 0.1% (w/v) DDM, 0.02% (w/v) CHS, 10% (v/v) glycerol, and 20 mM imidazole (wash buffer) and eluted in wash buffer supplemented with 230 mM imidazole. 2 mM CaCl₂ was then added to the eluate, which was further purified over the in-house anti-FLAG M1 affinity sepharose column. The resin was washed with HBS + 2 mM CaCl₂ supplemented with detergents, which was gradually exchanged from 0.1% DDM to 0.01% GDN. The receptor was eluted in HBS with 0.002% (w/v) GDN, 0.0002% (w/v) CHS, 0.2 mg/ml FLAG peptide and 5 mM EDTA, and further purified by a size-exclusion chromatography (SEC) column Superdex 200 10/300 GL equilibrated with SEC buffer comprised of HBS, 0.002% (w/v) GDN and 0.0002% (w/v) CHS.

The anti-BRIL Fab was expressed in E. coli and purified as described (Mukherjee et al., 2020). The anti-Fab Nb (Ereño-Orbea et al., 2018; Hermans et al., 2017) was cloned in pET26b+ vector with an N-terminal hexahistidine tag followed by a TEV protease site. The protein was expressed in E. coli BL21(DE3) cells and purified by Ni-NTA chromatography, followed by removal of the His-tag by TEV digestion. The final step in the purification was a SEC on a Superdex 75 prep grade column whereby monodisperse Nb was obtained. The purified receptor was incubated with 1.4-fold and 2-fold molar excess of anti-BRIL Fab and anti-Fab Nb, respectively, at 4°C for overnight. The mixture was concentrated to 500 µL and injected into a SEC column Superdex 200 10/300 GL equilibrated with the SEC buffer. The peak fractions were concentrated to ~2 mg/ml and 0.01% digitonin was added at this point. The solution was further concentrated to ~8 mg/ml for cryo-EM analysis.

To prepare the Wnt/Fzd complex, we expressed XWnt8 and hFzd5FL separately from Drosophila S2 cells or HEK293S GnTI^- cells, respectively. For solubilized XWnt8, we employed the XWnt8/mFzd8CRD co-expression system developed previously (Janda et al., 2012). Untagged XWnt8 and C-terminal Fc fusion mFzd8CRD were stably co-expressed in cell culture media, and secreted XWnt8/mFzd8CRD-Fc was captured on Protein A sepharose resin (Sigma). After washing the resin with HBS on column, only XWnt8 was eluted from the column with Wnt elution buffer containing 10 mM Hepes-Na pH 7.2, 500 mM NaCl and 0.1% DDM, in which DDM ‘wash out’ lipidated Wnt from CRD with competition. The eluted XWnt8 was then captured by ConA glycan affinity resin (Vector Laboratories) for detergent exchange. The detergent composition was gradually changed as follow; 0.1% DDM, 0.09% DDM/0.01% GDN, 0.05% DDM/0.05% GDN, 0.01% DDM/0.09% GDN, 0.1% GDN and 0.01% GDN. XWnt8 in GDN is eluted with HBS containing 0.01% GDN and 250 mM each Methyl α-D-glucopyranoside and Methyl α-D-mannopyranoside (Sigma). hFzd5FL was prepared by essentially the same scheme as hFzd5_ICL3BRIL. Briefly, wild-type hFzd5FL with N-terminal FLAG and C-terminal octahistidine tags was expressed on cell membrane using BacMam system, extracted in 1% DDM/0.2% CHS and first purified with Ni-NTA resin. Pool fractions of hFzd5FL from Ni-NTA resin in 0.1% DDM/0.02% CHS was further purified with anti-FLAG M1 affinity sepharose with detergent exchange to 0.01% GDN/0.002% CHS, and a Superdex 200 10/300 GL column equilibrated with the SEC buffer. 1.2 molar excess XWnt8 was added to the SEC purified hFzd5FL, and incubated at 4°C for overnight. The mixture was concentrated to 500 µL and injected into a Superdex 200 10/300 GL column equilibrated with the SEC buffer. The peak fractions were concentrated to 5–15 mg/ml with final <1 CMC (0.06%) digitonin for cryo-EM specimen preparations.

Cryo-EM specimen preparation and data collection

2.5 µL of either the hFzd5_ICL3BRIL/anti-BRIL Fab/anti-Fab Nb sample or the XWnt8/hFzd5FL sample was applied to glow-discharged 300 mesh gold grids (Quantifoil R1.2/1.3). Excess sample was blotted to a filter paper for 5 s before plunge-freezing using a Leica EM GP (Leica Microsystems) at 8°C and 90% humidity.

Cryo-EM data were collected at the CryoEM facility at the HHMI Janelia Research Campus. Images were collected on a Titan Krios operated at 300 kV equipped with a Gatan imaging filter and K3 camera in correlated double sampling super-resolution mode at a nominal magnification of 81,000x, corresponding to a physical pixel size of 1.078 Å. Each movie was recorded for a total of 6.125 s with 0.1225 s exposure per frame at an exposure rate of 9.5 electrons/pixel/second at the specimens, that yielded an electron counting of ~7.5 electrons/pixel/second at the camera. The datasets were collected using SerialEM (Mastronarde, 2005) software with the defocus range between −0.8 and −2.0 μm and beam-image shift to collect 9 movies from nine holes per stage shift and focusing.

Cryo-EM data processing

A total 10,898 movies were collected and subjected to beam-induced motion correction using MotionCor2 (Zheng et al., 2017) with binning to the physical pixel size. All subsequent data processing steps were performed with cryoSPARC (Punjani et al., 2017). Patched contrast transfer function parameters were estimated for each micrograph from the dose-weighted averages of all frames with default parameters. Initial auto-picking templates were generated after a round of reference-free picking on a subset of micrographs. A total of 6,483,398 ‘particles’ were picked with template-based auto picking. Several rounds of 2D classification resulted in 570,859 particles. Particles in this subgroup were subjected to several rounds of heterogeneous refinement and non-uniform refinement. A final subset of 207,021 particles were used for the final reconstruction. During the subsequent process, we noticed several ‘conformations’ were picked up in 3D classification attempts, as a hinge-like motion around the BRIL domain. The BRIL domain is notorious for its flexible nature, and is often poorly resolved or even disordered in crystallographic reconstructions. The final non-uniform refinement reconstruction yielded a structure at 4.6 Å resolution. The best final reconstruction (nominal resolution of 3.7 Å), was achieved with the full map fed into the ‘local-refinement’ routine, with a fulcrum on the BRIL domain. We attempted several ways of masking of individual parts, which did not result in a reconstruction as good as the using the full map including the detergent belt. The resolution of the final map was determined by gold standard Fourier shell correlation using the 0.143 criterion.

For the XWnt8/hFzd5FL dataset, a similar data processing approach was utilized. Initially, beam induced motion was corrected for 6,161 raw movies using MotionCor2. Dose-weighted micrographs were imported into cryoSPARC for all subsequent steps. The ‘Patch CTF’ routine was applied for CTF estimation, followed by template-free and template-based particle picking. A total of 6,431,867 ‘particles’ were picked for the final set, which underwent successive rounds of 2D classification to clean up the dataset. From 2D classes alone, it was already apparent that the XWnt8/hFzd5FL was highly dynamic and did not yield any discernible secondary structure features. The resulting 245,114 particles were further processed by ab-initio reconstructions and heterogeneous refinement in an attempt to further classify subsets. The final dataset of 33,149 particles was used for the 2D classification shown in Figure 1—figure supplement 5c and to generate a 3D reconstruction with unsatisfying quality.

Model building and refinement for hFzd5_ICL3BRIL/Fab/Nb

The homology model of hFzd5 was built on SWISS-MODEL server (swissmodel.expasy.org) using the crystal structures of rubredoxin fusion hFzd4TM (PDB ID: 6BD4) and BRIL fusion multi-domain Smo (PDB ID: 5L7D) as templates. The BRIL/Fab/Nb unit was modeled based on the completed Fab/Nb structure (PDB ID: 6ANI, and Hermans et al., 2017), and crystal structure of the BRIL/Fab complex (PDB ID: 6CBV). The hFzd5 and BRIL/Fab/Nb models are roughly fitted into the initial 4.6 Å map on UCSF Chimera. After real-space refinement with global minimization and rigid body constraints on Phenix (Adams et al., 2010), model was further fixed and refined iteratively using Coot (Emsley and Cowtan, 2004) for manual model building, inspection and correction, ISOLDE (Croll, 2018) on UCSF Chimera X (Goddard et al., 2018) for MD-based model idealization, and Phenix for real-space refinement. The map was updated to 3.9 Å then the final 3.7 Å during model building. The structure and map were visualized using UCSF Chimera X and PyMol (Schrödinger, LLC). All secondary structures in the figures were assigned using DSSP (Joosten et al., 2011). The cavities were detected using the CavityPlus server (Xu et al., 2018) with a default setting, and the dimensions of the cavities were calculated using ChExVis (Masood et al., 2015) server with the pocket residues assigned by CavityPlus as inputs and the probe size of 1 Å. For PDB deposition, the amino acid residues on hFzd5_ICL3BRIL were renumbered after the wwPDB validation step to separate chimeric BRIL protein from the hFzd5 region.

STF luciferase reporter assays

STF activity assays were performed using the embryonic kidney (HEK293T) cell line, with Fzd1, 2, 4, 5, 7 and 8 knockouts. The cell line is courtesy of Michael Boutros laboratory (Voloshanenko et al., 2017) and stably transfected with the 7TFP plasmid (Addgene plasmid #: 24308) encoding 7xTCF-Ffluc, termed 293-Fzd-KO-STF. 1−2 × 10⁶ cells in DMEM with 10% FBS were seeded into 6-well plates. After 24 hrs cells were transfected with 2 µg of pRK5 plasmid containing the coding region of human hFzd5 wild-type (Yu et al., 2012) or mutants using Lipofectamine 2000 (Thermo Fisher) according the manufacturer’s instruction. After 24 hr, cells were re-seeded in triplicate for each condition in 96-well plates, and after 20–24 hr of recovery, cells were stimulated with dilution series of recombinant Wnt3a (R and D) or Surrogate Wnt. The agonist concentrations were 0, 1.56, 6.25, 3.125 or 12.5 nM for Wnt3a and 0, 0.25, 1, 4 or 16 nM for surrogate Wnt. After 20–24 hr, cells were lysed in 30 µl 1x passive lysis buffer (Promega). 10 µl per well of lysate were assayed using the Luciferase assay kit (Promega) and luminance signal was measured using a SpectraMax plate reader. Data were analyzed and plotted in GraphPad Prism 7.

Expression analysis of Fzd5 wild-type and mutants in 293-Fzd-KO-STF

His-tagged monomeric DRPB_Fz8 was expressed and purified as previously described (Dang et al., 2019) and labelled with Alexa Fluor 647 NHS (Thermo Fisher) according manufacturer’s instruction. Approx. 1 × 10⁶ 293 Fzd-KO-STF cells were transiently transfected with pRK5 plasmids encoding human hFzd5 wild-type and mutants, and plated into 96-well V-bottom plates. Assay plates were centrifuged to pellet cells, cells were washed twice in FACS buffer (PBS with 1% BSA) and stained with DRPB_Fz8 for 1 hr at room temperature. Cells were washed twice and resuspended in FACS buffer and cell surface expression of hFzd5 wild-type and mutants by means of binding of DRPB_Fz8 was determined by fluorescence intensity analysis on an Accuri C6 flow cytometer (BD Biosciences). Histogram data were plotted in GraphPad Prism 7.

Acknowledgements

The cryo-EM data were collected at the Cryo-EM facility at the HHMI Janelia Research Campus; we thank Drs. Zhiheng Yu and Doreen Matthies for their generous supports in microscope operation and data collection. Preliminary cryo-EM screening is performed at the Cryo-EM Facility at the Department of Structural Biology, Stanford University and the Stanford-SLAC Cryo-EM facility; We thank Drs. Dong-Hua Chen and Elizabeth Montabana for their kind supports in operating the facilities and user training. Most of data were processed on the SLAC cluster; We thank Dr. Yee-Ting Li for support with computing resources. Dr. Satchal Erramilli assisted in generation of the anti-BRIL Fab. We thank Andrew Velasco for assistance, Caleb Glassman for scientific discussion.

This research was supported by NIH grants 1R01DK115728 to KCG, R01GM117372 and P50GM082545 to AAK, and by the Department of Energy, Laboratory Directed Research and Development program at SLAC National Accelerator Laboratory, under contract DE-AC02-76SF00515, to CG. KCG is an investigator of the Howard Hughes Medical Institute and the Younger Family Chair, and is supported by the Ludwig Institute. NT was supported in part by the Long-Term Fellowships from the Human Science Frontier Program Organization (LT000011/2016 L).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Cornelius Gati, Email: cgati@stanford.edu.

K Christopher Garcia, Email: kcgarcia@stanford.edu.

Yi Arial Zeng, Chinese Academy of Sciences, China.

Olga Boudker, Weill Cornell Medicine, United States.

Funding Information

This paper was supported by the following grants:

National Institutes of Health 1R01DK115728 to K Christopher Garcia.
Howard Hughes Medical Institute to K Christopher Garcia.
Ludwig Institute for Cancer Research to K Christopher Garcia.
National Institutes of Health R01GM117372 to Anthony A Kossiakoff.
National Institutes of Health P50GM082545 to Anthony A Kossiakoff.
U.S. Department of Energy DE-AC02-76SF00515 to Cornelius Gati.
Human Frontier Science Program LT000011/2016-L to Naotaka Tsutsumi.

Additional information

Competing interests

No competing interests declared.

KCG and CYJ are founders of Surrozen Therapeutics.

Author contributions

Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review and editing, Cryo-EM specimen preparation, screening and data collection, Model building and refinement with KMJ and CG.

Investigation, Writing - review and editing, Development of anti-BRIL Fab, and purification of the Fab and anti-Fab Nb under supervision of AAK.

Investigation.

Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - review and editing, Mutagenesis and cell signaling assay.

Formal analysis, Validation, Writing - review and editing, Model building and inspection.

Investigation, Writing - review and editing, Development and purification of the surrogate Wnt agonist under supervision of KCG.

Investigation.

Investigation, Preliminary cryo-EM data analysis (Krios XWnt8/hFzd5).

Resources, Supervision, Funding acquisition, Investigation, Writing - review and editing.

Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Writing - review and editing, Formal cryo-EM data analysis and map generation; Model building and inspection.

Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Project administration, Writing - review and editing.

Additional files

Transparent reporting form

elife-58464-transrepform.docx^{(67.3KB, docx)}

Data availability

The cryo-EM map has been deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-21927 and the model coordinate has been deposited in the Protein Data Bank (PDB) under accession number 6WW2. All cell-based assay data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2 (Figure 2 - Figure supplement 1) and Figure 4 in excel format. Material availability: The expression plasmid for the anti-BRIL Fab is available from AKK (koss@bsd.uchicago.edu) by request.

The following datasets were generated:

Tsutsumi N, Jude KM, Gati C, Garcia KC. 2020. Structure of human Frizzled5 by fiducial-assisted cryo-EM. Electron Microscopy Data Bank. EMD-21927

Tsutsumi N, Jude KM, Gati C, Garcia KC. 2020. Structure of human Frizzled5 by fiducial-assisted cryo-EM. RCSB Protein Data Bank. 6WW2

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eLife. doi: 10.7554/eLife.58464.sa1

Decision letter

Editor: Yi Arial Zeng¹

Reviewed by: Rami Hannoush²

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study reveals a new structural basis of Wnt signaling through Fzd and advances our mechanistic understanding on Wnt signaling transduction.

Decision letter after peer review:

Thank you for submitting your article "Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Olga Boudker as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Rami Hannoush (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The reviewers have judged that your manuscript is of interest, but as described below additional re-analyses are required before it is published. In particular, please pay attention to comments 1-2 by reviewer #2, concerns were raised regarding the authenticity of the ECL structures and heterodimerization mechanism.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Reviewer #1:

Tsutsumi et al. present the Cryo EM structure of FZD5, which they determined with the help of a rigid BRIL fusion in intracellular loop 3 and an anti-BRIL-Fab bound by an anti-Fab nanobody to help particle alignment. The structure shows clear density for the transmembrane, the BRIL, the Fab and the nanobody domains, but not for the FZD-CRD. Another attempt at solving a structure of FZD5 in complex with XWnt8 was unsuccessful due to inherent flexibility between the FZD TMD and the CRD-Wnt complex. The TMD of FZD5 shows a very narrow allosteric binding pocket as observed previously for FZD4. Taken together the authors hypothesize that it is very unlikely that Wnt ligands exert their function via the classic allosteric binding pocket of “normal” GPCR and propose a simple heterodimerization model with LRP receptors to activate β-catenin dependent Wnt signaling. To validate this model, they compare the signaling ability of FZD5 point mutants of the allosteric pocket induced by Wnt ligands or structurally unrelated synthetic Wnt surrogates.

This is a beautiful study and the results are interesting and advance our mechanistic understanding on Wnt signaling. The manuscript is well-written, the data presented in the study are of high quality and the conclusions are supported by the data presented.

Comments:

1) It would be useful to provide some more details about the constructs design. For example, how was the insertion of the BRIL domain optimized among loops? Why not fused as continuation of appropriate transmembrane helices? Did the insertion of the BRIL domain improve the hFzd5 stability?

2) Is it possible that the insertion of the BRIL domain (or together with Fab/Nb) can affect Fzd conformation or oligomeric state (because of its bulkiness)? It is also possible that different experimental conditions result in different oligomeric states of Fzd. The authors should comment on these points.

3) The signaling strength of low-expressed/unstable Y507A is similar to (or higher than) wild-type, which is not seen in K506A. Could the author clarify this observation from the structural point of view

4) The heterodimerization model provides valuable insight into the activation mechanism of canonical Wnt signaling. The data supports that allosteric activation via small ligands of the binding pocket in the center of the TM domain does not play a major role. However, it cannot fully be excluded, and though the authors state that this cannot be fully ruled out, this point needs to be emphasized in the paper.

5) This is the first structure of a FZD TM domain without any thermostabilizing mutations. It would be informative to comment on whether any structural differences between the FZD5 and FZD4 TM can directly be attributed to such mutations.

Reviewer #2:

General assessment

Considering the fundamental importance of Wnt signaling in basic biology and medicine, as well as the scarcity of the structural information about the core signaling components, the partial structure of Fz5 reported here merits publication in eLife. I have no problem with the structure of TM regions and the mechanistic interpretations built upon the structural as well as functional data of the mutants. However, I found that the structural analysis and conclusion about the extracellular parts are very crude and highly misleading. Either extensive re-analyses or omission of the data/arguments is strongly advised.

Substantive concerns

1) Authenticity of the ECL structures

In contrast to the TM region of Fz5 which showed high structural similarity with that of Fz4, the extracellular region comprising three ECLs and hinge differs significantly (Figure 2C). This region is of great biological importance because it is situated in a position that could structurally and functionally link ligand-binding CRD and the TM bundle, and therefore the accuracy of the model is critical. I found that the way authors present the loop structure (Figure 1—figure supplement 4B) is highly misleading. First of all, arbitrarily separating certain chunk of EM map and overlaying the model structure should be avoided, because there is no way of knowing which part of the molecule actually contributed to the observed volume. Using the data provided, I made volume map figure myself using Chimera after normalizing the data (mean=0, stdev=1) with the threshold of 3 σ. When viewed from different orientation, the quality of the fitting becomes unacceptably low, with the region near Arg601 having almost no density. I also have serious doubts about the disulfide bonding between C190 and C590 (mislabeled as C484 in the figure). If you look closely the alignment (Figure 1—figure supplement 1), C590-containing ECL3 invariably has two Cys in all Fz/Smo except for Fz4, which are disulfide-bonded in Smo. Connecting C590 with C190 would leave the surface-exposed C592 in an unpaired state. Considering the conservation across Fzs, C190 (C232 in Fz8) is most likely bonded to preceding C182 (C228 in Fz8). Therefore, the plausible interpretation would be C182-C190 and C590-C592. This is not trivial point, because it points to the possibility that the main-chain tracing of the current model could be completely wrong, which would undermine the overall discussion about the extracellular disulfide network.

2) Heterodimerization mechanism

The authors repeatedly mention, even in the article title itself, that ligand-induced heterodimerization is the mechanism responsible for the canonical Wnt signaling. I can agree that it does not conform to the allosteric mechanism found in class A GPCR, but the choice of word (heterodimerization) will cause great confusion/misunderstanding among readers, partly because the GPCR heterodimerization has also been postulated. I understand that the authors refer to the heterodimerization between Fz5 and LRP6, and that such interaction is undeniably critical based on the numerous published results as well as the authors' own surrogate agonist experiments. But authors failed to discuss equally-well documented mechanism of Fz homodimerization, which is not mutually exclusive with the heterodimer theory. Fz CRD alone is reported to undergo homodimerization (ref 17), so is Wnt3-CRD complex (ref 10). Probably the more important question to ask is whether the Fz-LRP heterodimer undergoes homodimer(or oligomer)ization. It is regrettable that the authors did not perform more extensive analysis on the di/oligomerization tendency of FL Fz5 before and after the Wnt8 binding. I would have liked it very much if statistical analysis on the dimer/monomer ratio found in the earlier SEC fractions were presented.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

In light of their advice I am delighted to say that we are happy, in principle, to publish a suitably revised version in eLife.

Please note that reviewer #2 commented on your revision and suggested a textual change in Abstract regarding the word "ligand-induced heterodimerization” and making appropriate adjustment in both Figure 1E and Figure 1—figure supplement 5E.

We therefore invite you to revise your paper one last time to edit your manuscript to comply with our format requirements and to maximise the accessibility and therefore the impact of your work.

Reviewer #1:

The authors have fully addressed my comments and this is an improved version of the paper that is ready for publication.

Reviewer #2:

The authors addressed the primary concern I expressed in the initial review (i.e., the authenticity of the ECL structure) by removing some segments from the model and accordingly modifying the figures. Overall, I think the paper is almost ready for the publication in eLife considering the fundamental importance of Wnt signaling in basic biology and medicine, as well as the scarcity of the structural information about the core signaling components. However, I could not find satisfying changes in the text regarding my second concern (i.e., clarification of the terminology of "heterodimerization mechanism").

1) Heterodimerization mechanism

Maybe my comment in the previous review was not clear. I was not suggesting the authors to emphasize the alternative signaling mechanism involving Fz-Fz homodimerization. My concern was the choice of the word (heterodimerization mechanism) because readers may get the wrong impression that they are claiming the GPCR (Fz) heterodimerization as the major signaling mechanism, particularly when readers only read Abstract. In the Abstract, I suggest changing "ligand-induced heterodimerization" to something like "Fzd/LRP6 heterodimerization".

2) EM images of Wnt8-Fz5

The authors revised the text to further emphasize the limitation of the data, which is fair and appropriate. And I can agree with their intention to keep the figure as the main figure. However, the newly added 3D cross-section of the Figure 1E is highly inappropriate. First of all, the 2D class averages of the original Figure 1E and Figure 1—figure supplement 5 are of low quality and can contain many misaligned particles. In general, calculating a 3D volume map does not make the data more reliable than calculating 2D averages (it is the opposite). More importantly, the "local refinement" feature in cryoSPARC program can only be performed after accurately determining the overall 3D structure, which is not the case here. This point is clearly mentioned in the developer's website (https://cryosparc.com/blog/local-refinement-snrnp-case-study) as "Before we can use any of the local refinement tools, we need an accurate picture of the overall structure…". Besides, there are no description about the mask they used in the local refinement nor the explanation of the heat map values. I strongly suggest authors to remove the "refined 3D map" for the Wnt-Fz complex from both Figure 1E and Figure 1—figure supplement 5E.

eLife. 2020 Aug 7;9:e58464. doi: 10.7554/eLife.58464.sa2

Author response

Reviewer #1:

Comments:

1) It would be useful to provide some more details about the constructs design. For example, how was the insertion of the BRIL domain optimized among loops? Why not fused as continuation of appropriate transmembrane helices? Did the insertion of the BRIL domain improve the hFzd5 stability?

As we elaborate in the Materials and methods Section, the BRIL domain is connected to TM5 and TM6 of hFzd5 by two short peptides (ARRQL and ARSTL) derived from TM5 and TM6 of A_2A adenosine receptor. TM5/6 of hFzd5, α-helix 1/4 of BRIL, and the two peptides formed continuous α-helices that permit cryo-EM particle alignment. In this study, we have not compared the stability of hFzd5_ICL3BRIL and hFzd5FL, for instance, by assessing the thermostability. However, we have noticed an overall 5-fold higher yield of the hFzd5_ICL3BRIL construct after expression and purification compared to hFzd5FL, suggesting superior stability. We have edited the Materials and methods section of the manuscript to address these points.

2) Is it possible that the insertion of the BRIL domain (or together with Fab/Nb) can affect Fzd conformation or oligomeric state (because of its bulkiness)? It is also possible that different experimental conditions result in different oligomeric states of Fzd. The authors should comment on these points.

We agree that we cannot entirely exclude that the BRIL insertion into the ICL3 of Fzd5 alters the propensity of Fzd5 for distinct conformational and oligomeric states. In reality, cryo-EM analysis and protein crystallization techniques both have aspects that can bias towards certain conformational states, but we are careful not to extend our interpretations beyond the most plausible explanations.

Concerning the oligomeric state, we predominantly observed peaks of monomeric hFzd5I_CL3BRIL and hFzd5FL on size-exclusion chromatography. The BRIL insertion does not noticeably change the preference for distinct oligomeric states. Also, to our knowledge, it has not been reported that the insertion of BRIL into the ICL3 of GPCRs triggers changes to their oligomeric states. Hence, we focus our discussion on the description of the oligomeric states without extending our speculations on their functional implications.

3) The signaling strength of low-expressed/unstable Y507A is similar to (or higher than) wild-type, which is not seen in K506A. Could the author clarify this observation from the structural point of view

We thank the reviewer for this comment. Our flow cytometry data show that the K506A variant has absolutely no detectable expression, whereas the Y507A variant has a relatively low cell surface expression that was enough to saturate the Wnt3a-dependent signaling. This suggests while the Y507A mutation impairs expression, the K506A is detrimental to the stability of hFzd5. Among ten Frizzled family members, K^7.41 is 100% conserved where the sidechain plays a role in the inter-helical packing between TM6 and TM7 as mentioned, by bridging Y^6.51 (90% conserved, L for Fzd8), E^6.54 (100% conserved) and Y^7.42 (70% conserved, I for Fzd4/9/10) in the structure, which suggests its functional importance. We edited the corresponding discussion lightly to imply the class-conserved K506 is more important than Y507.

4) The heterodimerization model provides valuable insight into the activation mechanism of canonical Wnt signaling. The data supports that allosteric activation via small ligands of the binding pocket in the center of the TM domain does not play a major role. However, it cannot fully be excluded, and though the authors state that this cannot be fully ruled out, this point needs to be emphasized in the paper.

We agree that although the close packing of the TMs suggests that the pocket of the receptor occludes small molecules, we cannot fully exclude that certain classes of small molecules can be accommodated in the “ligand-binding” pocket. In fact, we have cited the publication reporting the Smo agonist SAG1.3 could act as a partial agonist for Fzd6-Gi signaling. This is in good agreement with our existing discussion regarding the mechanism of Fzd receptor activation.

Our data suggest that simple cross-linking of Fzd and LRP6 can trigger the intracellular β-catenin signaling, as the surrogate agonists have shown. This mechanism is not mutually exclusive with alternative mechanisms to activate intracellular signaling pathways that might be triggered by small-molecule ligands. Hence, we further interpret that the conformational change model might be more applicable to intracellular signaling mediated by G proteins. Fzd-dependent G protein signaling might be more sensitive to conformational change. The possibility of conformational change during G protein signaling has been included in the current revised version.

5) This is the first structure of a FZD TM domain without any thermostabilizing mutations. It would be informative to comment on whether any structural differences between the FZD5 and FZD4 TM can directly be attributed to such mutations.

The stabilizing mutations of Fzd4 were designed by the group of Prof. Xu based on multiple sequence alignment of Fzd members. For three of the four mutations, amino acids that occur naturally in Fzd5 were chosen (C450I, C507F, and S508Y for the Fzd4 structure). The remaining one is also similar (M309L for the Fzd4 structure, and Fzd5 has V at the corresponding position). Based on this analysis, we hypothesize that Fzd5 is perhaps naturally more stable compared to Fzd4. Given these similarities, we cannot draw any meaningful conclusion by comparing variations at these particular sites.

Reviewer #2:

Substantive concerns

1) Authenticity of the ECL structures

In contrast to the TM region of Fz5 which showed high structural similarity with that of Fz4, the extracellular region comprising three ECLs and hinge differs significantly (Figure 2C). This region is of great biological importance because it is situated in a position that could structurally and functionally link ligand-binding CRD and the TM bundle, and therefore the accuracy of the model is critical. I found that the way authors present the loop structure (Figure 1—figure supplement 4B) is highly misleading. First of all, arbitrarily separating certain chunk of EM map and overlaying the model structure should be avoided, because there is no way of knowing which part of the molecule actually contributed to the observed volume. Using the data provided, I made volume map figure myself using Chimera after normalizing the data (mean=0, stdev=1) with the threshold of 3 σ. When viewed from different orientation, the quality of the fitting becomes unacceptably low, with the region near Arg601 having almost no density. I also have serious doubts about the disulfide bonding between C190 and C590 (mislabeled as C484 in the figure). If you look closely the alignment (Figure 1—figure supplement 1), C590-containing ECL3 invariably has two Cys in all Fz/Smo except for Fz4, which are disulfide-bonded in Smo. Connecting C590 with C190 would leave the surface-exposed C592 in an unpaired state. Considering the conservation across Fzs, C190 (C232 in Fz8) is most likely bonded to preceding C182 (C228 in Fz8). Therefore, the plausible interpretation would be C182-C190 and C590-C592. This is not trivial point, because it points to the possibility that the main-chain tracing of the current model could be completely wrong, which would undermine the overall discussion about the extracellular disulfide network.

We agree with the reviewer that our initial model was not sufficiently conservative. Our interpretation of the EM volume was C182(unmodeled)-C486/C190-C484, however, we also made variants with C182(unmodeled)-C190/C484-C486 during our model building attempts, and we agree that it is not appropriate to judge the disulfide bonding network based on this map.

Based on the reviewer’s suggestions, we have made the following adjustments.

1) We have removed residues P487 to P498, which includes the flexible region in ICL3 and R495, from our model. We agree that the EM volume of this region is ambiguous. Although ICL3 likely adopts multiple conformations, including the one we have previously modeled, we recognize that it implies false precision when a single model is placed (subsection “Structural comparison between hFzd5, hFzd4 and hSmo”).

2) We have removed residues F188 to C192 and the sidechain of C484 from our model. This is to avoid misleading conclusions about the most flexible yet important regions of the structure. Further, we have revised the Discussion in our manuscript accordingly. We note that the hinge domain and ICL3 of the Fzd5/8 subfamily have a unique distribution of cysteine residues among the class F GPCRs, and wish to perform additional experiments to more adequately address this question.

Further, we apologize for the inconsistency in residues numbering. In the initial submission, we provided the coordinates directly from Phenix refine. Here, we have attached a revised model with renumbered residues, so it is consistent with the figures.

2) Heterodimerization mechanism

The authors repeatedly mention, even in the article title itself, that ligand-induced heterodimerization is the mechanism responsible for the canonical Wnt signaling. I can agree that it does not conform to the allosteric mechanism found in class A GPCR, but the choice of word (heterodimerization) will cause great confusion/misunderstanding among readers, partly because the GPCR heterodimerization has also been postulated. I understand that the authors refer to the heterodimerization between Fz5 and LRP6, and that such interaction is undeniably critical based on the numerous published results as well as the authors' own surrogate agonist experiments. But authors failed to discuss equally-well documented mechanism of Fz homodimerization, which is not mutually exclusive with the heterodimer theory. Fz CRD alone is reported to undergo homodimerization (ref 17), so is Wnt3-CRD complex (ref 10). Probably the more important question to ask is whether the Fz-LRP heterodimer undergoes homodimer(or oligomer)ization. It is regrettable that the authors did not perform more extensive analysis on the di/oligomerization tendency of FL Fz5 before and after the Wnt8 binding. I would have liked it very much if statistical analysis on the dimer/monomer ratio found in the earlier SEC fractions were presented.

In several crystal structures, the isolated Fzd-CRDs in complex with lipids or Wnt3a were observed in dimeric arrangements, and we have cited the corresponding publications. However, we have previously carried out single-molecule TIRF microscopy imaging of full-length Fzd8 and LRP6 on live cell membranes with and without Wnt (Janda et al., 2017). We have found that the predominant state of the signaling complexes is a 1:1 monomer of the Fzd8/LRP6 heterodimer, with less populated higher oligomeric states. We have not seen evidence of Fzd homodimers on cells, although this issue remains to be fully resolved. We have also observed and described a small population of hFzd5_ICL3BRIL/Fab/Nb dimers in the cryo-EM 2D class averages. Given the published data in hand, we feel that we have given a fair and transparent account of this issue and do not think that anything else is within the scope of the current paper.

We also note that purified hFzd5FL and hFzd5_ICL3BRIL in detergent micelles elute from size-exclusion chromatography (SEC) at volumes predictive of monomers. The addition of XWnt8 only leads to a marginal faster migration through the size-exclusion column, suggesting a 1:1 complex formation. However, the SEC profiles also show some higher molecular weight shoulders. These are very common for most other GPCRs we have purified, and the amount of the small shoulder varies between experiments and protein preparations. As we cannot know whether it represents the physiological dimer (multimer) or artifacts (such as misfolded and aggregated receptors, and unnatural head-to-tail dimers often observed for detergent-solubilized membrane proteins), we are hesitant to quantify the ratio based on the unclear SEC observation that can lead to erroneous conclusions.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #2:

The authors addressed the primary concern I expressed in the initial review (i.e., the authenticity of the ECL structure) by removing some segments from the model and accordingly modifying the figures. Overall, I think the paper is almost ready for the publication in eLife considering the fundamental importance of Wnt signaling in basic biology and medicine, as well as the scarcity of the structural information about the core signaling components. However, I could not find satisfying changes in the text regarding my second concern (i.e., clarification of the terminology of "heterodimerization mechanism").

1) Heterodimerization mechanism

Maybe my comment in the previous review was not clear. I was not suggesting the authors to emphasize the alternative signaling mechanism involving Fz-Fz homodimerization. My concern was the choice of the word (heterodimerization mechanism) because readers may get the wrong impression that they are claiming the GPCR (Fz) heterodimerization as the major signaling mechanism, particularly when readers only read Abstract. In the Abstract, I suggest changing "ligand-induced heterodimerization" to something like "Fzd/LRP6 heterodimerization".

As suggested by the reviewer, we revised the word in the Abstract and the impact statement.

2) EM images of Wnt8-Fz5

The authors revised the text to further emphasize the limitation of the data, which is fair and appropriate. And I can agree with their intention to keep the figure as the main figure. However, the newly added 3D cross-section of the Figure 1E is highly inappropriate. First of all, the 2D class averages of the original Figure 1E and Figure 1—figure supplement 5 are of low quality and can contain many misaligned particles. In general, calculating a 3D volume map does not make the data more reliable than calculating 2D averages (it is the opposite). More importantly, the "local refinement" feature in cryoSPARC program can only be performed after accurately determining the overall 3D structure, which is not the case here. This point is clearly mentioned in the developer's website (https://cryosparc.com/blog/local-refinement-snrnp-case-study) as "Before we can use any of the local refinement tools, we need an accurate picture of the overall structure…". Besides, there are no description about the mask they used in the local refinement nor the explanation of the heat map values. I strongly suggest authors to remove the "refined 3D map" for the Wnt-Fz complex from both Figure 1E and Figure 1—figure supplement 5E.

As suggested by the reviewer, we deleted all data regarding the attempts at 3D refinement of XWnt8/hFz5FL.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

Tsutsumi N, Jude KM, Gati C, Garcia KC. 2020. Structure of human Frizzled5 by fiducial-assisted cryo-EM. Electron Microscopy Data Bank. EMD-21927 [DOI] [PMC free article] [PubMed]
Tsutsumi N, Jude KM, Gati C, Garcia KC. 2020. Structure of human Frizzled5 by fiducial-assisted cryo-EM. RCSB Protein Data Bank. 6WW2 [DOI] [PMC free article] [PubMed]

Supplementary Materials

Figure 2—figure supplement 1—source data 1. Raw FACS histogram data for Figure 2—figure supplement 1 showing cell-surface expression of hFzd5 variants.

elife-58464-fig2-figsupp1-data1.xlsx^{(23.9KB, xlsx)}

Figure 4—source data 1. Raw data points for the signaling experiments shown as circles in Figure 4.

elife-58464-fig4-data1.xlsx^{(13KB, xlsx)}

Transparent reporting form

elife-58464-transrepform.docx^{(67.3KB, docx)}

Data Availability Statement

The following datasets were generated:

Tsutsumi N, Jude KM, Gati C, Garcia KC. 2020. Structure of human Frizzled5 by fiducial-assisted cryo-EM. Electron Microscopy Data Bank. EMD-21927

Tsutsumi N, Jude KM, Gati C, Garcia KC. 2020. Structure of human Frizzled5 by fiducial-assisted cryo-EM. RCSB Protein Data Bank. 6WW2

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PERMALINK

Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling

Naotaka Tsutsumi

Somnath Mukherjee

Deepa Waghray

Claudia Y Janda

Kevin M Jude

Yi Miao

John S Burg

Nanda Gowtham Aduri

Anthony A Kossiakoff

Cornelius Gati

K Christopher Garcia

Roles

Abstract

Introduction

Results and discussion

Structure determination and analysis

Figure 1. Design scheme and cryo-EM analysis of hFzd5.

Figure 1—figure supplement 1. Multiple sequence alignment of human class F GPCRs.

Figure 1—figure supplement 2. Purification, data collection and 2D classification of hFzd5ICL3BRIL/Fab/Nb.

Figure 1—figure supplement 3. Cryo-EM data analysis of hFzd5ICL3BRIL/Fab/Nb.

Figure 1—figure supplement 4. Cryo-EM map of representative built in model.

Figure 1—figure supplement 5. Purification, data collection and 2D class averages of the XWnt8/hFzd5FL complex.

Table 1. Cryo-EM data collection and refinement statistics.

Structural comparison between hFzd5, hFzd4 and hSmo

Figure 2. Overall structure of hFzd5 and structural comparison with hFzd4 and hSmo.

Figure 2—figure supplement 1. Confirmation of hFzd5 receptor expression in HEK293T transfected cells.

Figure 3. Conformational differences between hFzd5, hFzd4 and hSmo.

Probing the effect of structure-guided hFzd5 mutations on signaling

Figure 4. Structure-guided hFzd5 mutant signaling assays.

Materials and methods

Key resources table.

Cell lines

Protein expression and purification for cryo-EM study

Cryo-EM specimen preparation and data collection

Cryo-EM data processing

Model building and refinement for hFzd5ICL3BRIL/Fab/Nb

STF luciferase reporter assays

Expression analysis of Fzd5 wild-type and mutants in 293-Fzd-KO-STF

Acknowledgements

Funding Statement

Contributor Information

Funding Information

Additional information

Competing interests

Author contributions

Additional files

Data availability

References

Decision letter

Roles

Author response

Associated Data

Data Citations

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Figure 1—figure supplement 2. Purification, data collection and 2D classification of hFzd5_ICL3BRIL/Fab/Nb.

Figure 1—figure supplement 3. Cryo-EM data analysis of hFzd5_ICL3BRIL/Fab/Nb.

Model building and refinement for hFzd5_ICL3BRIL/Fab/Nb