Beyond ß-catenin: prospects for a larger catenin network in the nucleus.

ß-catenin is widely regarded as the primary transducer of canonical WNT signals to the nucleus. In most vertebrates, there are eight additional catenins that are structurally related to ß-catenin, and three α-catenin genes encoding actin-binding proteins that are structurally related to vinculin. Although these catenins were initially identified in association with cadherins at cell-cell junctions, more recent evidence suggests that the majority of catenins also localize to the nucleus and regulate gene expression. Moreover, the number of catenins reported to be responsive to canonical WNT signals is increasing. Here, we posit that multiple catenins form a functional network in the nucleus, possibly engaging in conserved protein-protein interactions that are currently better characterized in the context of actin-based cell junctions.

Regulation of stem cell identity by miR-200a during spinal cord regeneration.

Axolotls are an important model organism for multiple types of regeneration, including functional spinal cord regeneration. Remarkably, axolotls can repair their spinal cord after a small lesion injury and can also regenerate their entire tail following amputation. Several classical signaling pathways that are used during development are reactivated during regeneration, but how this is regulated remains a mystery. We have previously identified miR-200a as a key factor that promotes successful spinal cord regeneration. Here, using RNA-seq analysis, we discovered that the inhibition of miR-200a results in an upregulation of the classical mesodermal marker brachyury in spinal cord cells after injury. However, these cells still express the neural stem cell marker sox2. In vivo cell tracking allowed us to determine that these cells can give rise to cells of both the neural and mesoderm lineage. Additionally, we found that miR-200a can directly regulate brachyury via a seed sequence in the 3'UTR of the gene. Our data indicate that miR-200a represses mesodermal cell fate after a small lesion injury in the spinal cord when only glial cells and neurons need to be replaced.

HCMV UL8 interaction with ß-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

IMPORTANCE: CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/ß-catenin pathway as an important component of viral reactivation. We further define that pUL8 and ß-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with ß-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

Unraveling the folding-assisted unbinding mechanism of TCF with its binding partner ß-catenin.

This study utilizes molecular dynamics simulations aided with multiple walker parallel bias metadynamics to investigate the TCF unbinding mechanism from the ß-catenin interface. The results, consistent with experimental binding affinity calculations, unveil a folding-assisted unbinding mechanism.

Wnt Signalosome Assembly by DEP Domain Swapping of Dishevelled.

Extracellular signals are often transduced by dynamic signaling complexes ("signalosomes") assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of ß-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to ß-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.

Diffusion of a disordered protein on its folded ligand.

Intrinsically disordered proteins often form dynamic complexes with their ligands. Yet, the speed and amplitude of these motions are hidden in classical binding kinetics. Here, we directly measure the dynamics in an exceptionally mobile, high-affinity complex. We show that the disordered tail of the cell adhesion protein E-cadherin dynamically samples a large surface area of the protooncogene ß-catenin. Single-molecule experiments and molecular simulations resolve these motions with high resolution in space and time. Contacts break and form within hundreds of microseconds without a dissociation of the complex. The energy landscape of this complex is rugged with many small barriers (3 to 4 kBT) and reconciles specificity, high affinity, and extreme disorder. A few persistent contacts provide specificity, whereas unspecific interactions boost affinity.

Structural optimization of novel Ras modulator for treatment of Colorectal cancer by promoting ß-catenin and Ras degradation.

Ras protein has been considered a fascinating target for anticancer therapy because its malfunction is closely related to cancer. However, Ras has been considered undruggable because of the failure to regulate its malfunction by controlling the Ras activation mechanism. Recently, Lumakras targeting the G12C mutation was approved, and therapeutic interest in Ras for anticancer therapy has been rejuvenated. Here, we present a series of compounds that inhibit Ras via a unique mechanism of action that exploits the relationship between the Wnt/ß-catenin pathway and Ras. KYA1797K (1) binds to axin to stabilize the ß-catenin destruction complex that causes the phosphorylation and subsequent degradation of Ras, similar to canonical ß-catenin regulation. Based on the chemical structure of 1, we performed a structural optimization and identified 3-(2-hydroxyethyl)-5-((6-(4-nitrophenyl)pyridin-2-yl)methylene)thiazolidine-2,4-dione (13d) as the most potent compound. 13d displayed antitumor effects in a colorectal cancer model with enhanced inhibition activity on Ras. The results of this study suggest that the further development of 13d could contribute to the development of Ras inhibitors with novel mechanisms of action.

PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

Poricoic acid A suppresses renal fibroblast activation and interstitial fibrosis in UUO rats via upregulating Sirt3 and promoting ß-catenin K49 deacetylation.

Renal interstitial fibrosis is the common pathological process of various chronic kidney diseases to end-stage renal disease. Inhibition of fibroblast activation attenuates renal interstitial fibrosis. Our previous studies show that poricoic acid A (PAA) isolated from Poria cocos is a potent anti-fibrotic agent. In the present study we investigated the effects of PAA on renal fibroblast activation and interstitial fibrosis and the underlying mechanisms. Renal interstitial fibrosis was induced in rats or mice by unilateral ureteral obstruction (UUO). UUO rats were administered PAA (10 mg·kg-1·d-1, i.g.) for 1 or 2 weeks. An in vitro model of renal fibrosis was established in normal renal kidney fibroblasts (NRK-49F cells) treated with TGF-ß1. We showed that PAA treatment rescued Sirt3 expression, and significantly attenuated renal fibroblast activation and interstitial fibrosis in both the in vivo and in vitro models. In TGF-ß1-treated NRK-49F cells, we demonstrated that Sirt3 deacetylated ß-catenin (a key transcription factor of fibroblast activation) and then accelerated its ubiquitin-dependent degradation, thus suppressing the protein expression and promoter activity of pro-fibrotic downstream target genes (twist, snail1, MMP-7 and PAI-1) to alleviate fibroblast activation; the lysine-49 (K49) of ß-catenin was responsible for Sirt3-mediated ß-catenin deacetylation. In molecular docking analysis, we found the potential interaction of Sirt3 and PAA. In both in vivo and in vitro models, pharmacological activation of Sirt3 by PAA significantly suppressed renal fibroblast activation via facilitating ß-catenin K49 deacetylation. In UUO mice and NRK-49F cells, Sirt3 overexpression enhanced the anti-fibrotic effect of PAA, whereas Sirt3 knockdown weakened the effect. Taken together, PAA attenuates renal fibroblast activation and interstitial fibrosis by upregulating Sirt3 and inducing ß-catenin K49 deacetylation, highlighting Sirt3 functions as a promising therapeutic target of renal fibroblast activation and interstitial fibrosis.

Phosphorylation of human CEACAM1-LF by PKA and GSK3ß promotes its interaction with ß-catenin.

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for ß-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the 15N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bß but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3ß binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3ß able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of ß-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3ß may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with ß-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.

An ensemble of flexible conformations underlies mechanotransduction by the cadherin-catenin adhesion complex.

The cadherin-catenin adhesion complex is the central component of the cell-cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•ß-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and ß-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of ß-catenin forms a flexible "tongue" that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin-catenin adhesion complex in mechanotransduction.

Epithelial-specific isoforms of protein 4.1R promote adherens junction assembly in maturing epithelia.

Epithelial adherens junctions (AJs) and tight junctions (TJs) undergo disassembly and reassembly during morphogenesis and pathological states. The membrane-cytoskeleton interface plays a crucial role in junctional reorganization. Protein 4.1R (4.1R), expressed as a diverse array of spliceoforms, has been implicated in linking the AJ and TJ complex to the cytoskeleton. However, which specific 4.1 isoform(s) participate and the mechanisms involved in junctional stability or remodeling remain unclear. We now describe a role for epithelial-specific isoforms containing exon 17b and excluding exon 16 4.1R (4.1R+17b) in AJs. 4.1R+17b is exclusively co-localized with the AJs. 4.1R+17b binds to the armadillo repeats 1-2 of ß-catenin via its membrane-binding domain. This complex is linked to the actin cytoskeleton via a bispecific interaction with an exon 17b-encoded peptide. Exon 17b peptides also promote fodrin-actin complex formation. Expression of 4.1R+17b forms does not disrupt the junctional cytoskeleton and AJs during the steady-state or calcium-dependent AJ reassembly. Overexpression of 4.1R-17b forms, which displace the endogenous 4.1R+17b forms at the AJs, as well as depletion of the 4.1R+17b forms both decrease junctional actin and attenuate the recruitment of spectrin to the AJs and also reduce E-cadherin during the initial junctional formation of the AJ reassembly process. Expressing 4.1R+17b forms in depleted cells rescues junctional localization of actin, spectrin, and E-cadherin assembly at the AJs. Together, our results identify a critical role for 4.1R+17b forms in AJ assembly and offer additional insights into the spectrin-actin-4.1R-based membrane skeleton as an emerging regulator of epithelial integrity and remodeling.

Exploring the structural significance of molecular switch mechanism alias motif phosphorylation in Wnt/ß-catenin and their crucial role in triple-negative breast cancer.

ß-Catenin, a key transcriptional factor involved in the canonical Wnt signaling pathway, is regulated by a cascade of phosphorylations and plays a major role in the progression of triple-negative breast cancer (TNBC). However, the phosphorylation induced conformational changes in a ß-Catenin is still poorly understood. Hence, we adopted a conventional molecular dynamics approach to study phosphorylations present in a sequence motif Ser 552 675 and Tyr670 of the ß-Catenin domain and analyzed in terms of structural transitions, bond formation, and folding-misfolding conformations. Our results unveil the ß-Catenin linear motif 549-555 (RRTSMGG) of armadillo repeats domain prefers order to disorder state. In contrast, helix C associated with 670-678 (YKKRLSVEL) motif prefers disorder to order upon phosphorylation of Ser 552 675 and Tyr670. In addition, the crucial secondary structural transition from α-helix to coil induced by phospho Ser552 and phospho Tyr670 of ß-Catenin ARM domain connecting helix C modifies conformational diversity and binding affinities of the complex interaction in functional regulation significantly. Moreover, the post phosphorylation disrupted the hydrogen bond interactions (Ser552-Arg549, Arg550-Asp546 and Ser675-Lys672) and abolished the residual alliance with hydrophobic interactions (Tyr670-Leu674) that easily interrupt in secondary structure packing as well as folding conformations connecting ARM and helix C (R10, 12 & R1C) compared to unphosphorylation. Our integrated computational analysis may help in shedding light on understanding the induced folding and unfolding pattern due to motif phosphorylations. Overall, our results provide an atomistic structural description of the way phosphorylation facilitates conformational and dynamic changes in ß-Catenin, a fundamental molecular switch mechanism in triple-negative breast cancer pathogenesis.

Cytosolic ß-catenin is involved in macrophage M2 activation and antiviral defense in teleosts: Delineation through molecular characterization of ß-catenin homolog from redlip mullet (Planiliza haematocheila).

ß-catenin is a structural protein that makes the cell-cell connection in adherence junctions. Besides the structural functions, it also plays a role as a central transducer of the canonical Wnt signaling cascade, regulating nearly four hundred genes related to various cellular processes. Recently the immune functions of ß-catenin during pathogenic invasion have gained more attention. In the present study, we elucidated the immune function of fish ß-catenin by identifying and characterizing the ß-catenin homolog (PhCatß) from redlip mullet, Planiliza haematocheila. The complete open reading frame of PhCatß consists of 2352 bp, which encodes a putative ß-catenin homolog (molecular weight: 85.7 kDa). Multiple sequence alignment analysis revealed that ß-catenin is highly conserved in vertebrates. Phylogenetic reconstruction demonstrated the close evolutionary relationship between PhCatß and other fish ß-catenin counterparts. The tissue distribution analysis showed the highest mRNA expression of PhCatß in heart tissues of the redlip mullet under normal physiological conditions. While in response to pathogenic stress, the PhCatß transcription level was dramatically increased in the spleen and gill tissues. The overexpression of PhCatß stimulated M2 polarization and cell proliferation of murine RAW 264.7 macrophage. In fish cells, the overexpression of PhCatß resulted in a significant upregulation of antiviral gene transcription and vice versa. Moreover, the overexpression of PhCatß could inhibit the replication of VHSV in FHM cells. Our results strongly suggest that PhCatß plays a role in macrophage activation and antiviral immune response in redlip mullet.

The 15-Amino Acid Repeat Region of Adenomatous Polyposis Coli Is Intrinsically Disordered and Retains Conformational Flexibility upon Binding ß-Catenin.

The tumor suppressor Adenomatous polyposis coli (APC) is a large, multidomain protein with many identified cellular functions. The best characterized role of APC is to scaffold a protein complex that negatively regulates Wnt signaling via ß-catenin destruction. This destruction is mediated by ß-catenin binding to centrally located 15- and 20-amino acid repeat regions of APC. More than 80% of cancers of the colon and rectum present with an APC mutation. Most carcinomas with mutant APC express a truncated APC protein that retains the ∼200-amino acid long' 15-amino acid repeat region'. This study demonstrates that the 15-amino acid repeat region of APC is intrinsically disordered. We investigated the backbone dynamics in the presence of ß-catenin and predicted residues that may contribute to transient secondary features. This study reveals that the 15-amino acid region of APC retains flexibility upon binding ß-catenin and that APC does not have a single, observable "highest-affinity" binding site for ß-catenin. This flexibility potentially allows ß-catenin to be more readily captured by APC and then remain accessible to other elements of the destruction complex for subsequent processing.

Recognition of physiological phosphorylation sites by p21-activated kinase 4.

Many serine/threonine protein kinases discriminate between serine and threonine substrates as a filter to control signaling output. Among these, the p21-activated kinase (PAK) group strongly favors phosphorylation of Ser over Thr residues. PAK4, a group II PAK, almost exclusively phosphorylates its substrates on serine residues. The only well documented exception is LIM domain kinase 1 (LIMK1), which is phosphorylated on an activation loop threonine (Thr508) to promote its catalytic activity. To understand the molecular and kinetic basis for PAK4 substrate selectivity we compared its mode of recognition of LIMK1 (Thr508) with that of a known serine substrate, ß-catenin (Ser675). We determined X-ray crystal structures of PAK4 in complex with synthetic peptides corresponding to its phosphorylation sites in LIMK1 and ß-catenin to 1.9 Å and 2.2 Å resolution, respectively. We found that the PAK4 DFG + 1 residue, a key determinant of phosphoacceptor preference, adopts a sub-optimal orientation when bound to LIMK1 compared to ß-catenin. In peptide kinase activity assays, we find that phosphoacceptor identity impacts catalytic efficiency but does not affect the Km value for both phosphorylation sites. Although catalytic efficiency of wild-type LIMK1 and ß-catenin are equivalent, T508S mutation of LIMK1 creates a highly efficient substrate. These results suggest suboptimal phosphorylation of LIMK1 as a mechanism for controlling the dynamics of substrate phosphorylation by PAK4.

An engineered antibody fragment targeting mutant ß-catenin via major histocompatibility complex I neoantigen presentation.

Mutations in CTNNB1, the gene encoding ß-catenin, are common in colon and liver cancers, the most frequent mutation affecting Ser-45 in ß-catenin. Peptides derived from WT ß-catenin have previously been shown to be presented on the cell surface as part of major histocompatibility complex (MHC) class I, suggesting an opportunity for targeting this common driver gene mutation with antibody-based therapies. Here, crystal structures of both the WT and S45F mutant peptide bound to HLA-A*03:01 at 2.20 and 2.45 Å resolutions, respectively, confirmed the accessibility of the phenylalanine residue for antibody recognition. Phage display was then used to identify single-chain variable fragment clones that selectively bind the S45F mutant peptide presented in HLA-A*03:01 and have minimal WT or other off-target binding. Following the initial characterization of five clones, we selected a single clone, E10, for further investigation. We developed a computational model of the binding of E10 to the mutant peptide-bound HLA-A3, incorporating data from affinity maturation as initial validation. In the future, our model may be used to design clones with maintained specificity and higher affinity. Such derivatives could be adapted into either cell-based (CAR-T) or protein-based (bispecific T-cell engagers) therapies to target cancer cells harboring the S45F mutation in CTNNB1.

ß-Catenin-RAS interaction serves as a molecular switch for RAS degradation via GSK3ß.

RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/ß-catenin pathway, and glycogen synthase kinase 3 beta (GSK3ß) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3ß-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) ß-catenin. Here, we show that ß-catenin directly interacts with RAS at the α-interface region that contains the GSK3ß phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior ß-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the ß-catenin-RAS interaction by binding to ß-catenin rescues the GSK3ß-mediated RAS degradation in colorectal cancer (CRC) cells that express MT ß-catenin. The coregulation of ß-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/ß-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.

Targeted disruption of the BCL9/ß-catenin interaction by endosomal-escapable nanoparticles functionalized with an E-cadherin-derived peptide.

Abnormal activation of the Wnt/ß-catenin signaling pathway, which underlies multiple malignancies, promotes tumor progression; drugs that can block this pathway are therefore highly attractive candidates for anticancer therapy. Using a therapeutic peptide derived from E-cadherin region V (cECRV), we sought to develop a potent and selective antagonist of ß-catenin that can disrupt the carcinogenic interaction between ß-catenin and BCL9. More importantly, to overcome the pharmacological obstacles of peptide-derived therapeutics (poor nuclease stability and low membrane permeability), a gold nanoparticle (AuNP)-based nanocarrier was designed to deliver cECRV into the cytoplasm to modulate the intracellular interaction of ß-catenin and BCL9. The resultant nanoparticle, pAuNP-cECRV, showed no cytotoxicity towards normal peripheral blood mononuclear cells and induced cycle arrest and subsequent apoptosis of Wnt-hyperactive cancer cells by antagonizing ß-catenin to inhibit the Wnt pathway. Our results indicate that pAuNP-cECRV is very promising for application as an efficient and safe peptide delivery vector for cancer therapy.

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding.

Frizzled (FZD) receptors mediate Wnt signaling in diverse processes ranging from bone growth to stem cell activity. Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors. Despite the progress in biochemical understanding of Wnt-FZD receptor interactions, the molecular basis for recognition of Wnt cis-unsaturated fatty acyl groups by the cysteine-rich domain (CRD) of FZD receptors remains elusive. Here, we determined a crystal structure of human FZD7 CRD unexpectedly bound to a 24-carbon fatty acid. We also report a crystal structure of human FZD5 CRD bound to C16:1 cis-Δ9 unsaturated fatty acid. Both structures reveal a dimeric arrangement of the CRD. The lipid-binding groove exhibits flexibility and spans both monomers, adopting a U-shaped geometry that accommodates the fatty acid. Re-evaluation of the published mouse FZD8 CRD structure reveals that it also shares the same architecture as FZD5 and FZD7 CRDs. Our results define a common molecular mechanism for recognition of the cis-unsaturated fatty acyl group, a necessary posttranslational modification of Wnts, by multiple FZD receptors. The fatty acid bridges two CRD monomers, implying that Wnt binding mediates FZD receptor dimerization. Our data uncover possibilities for the arrangement of Wnt-FZD CRD complexes and shed structural insights that could aide in the identification of pharmacological strategies to modulate FZD receptor function.