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

Abstract

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.nt signaling is evolutionarily conserved from metazoans to humans (1). It is critical for tissue homeostasis and during development (2, 3). On the other hand, derangements in Wnt signaling are associated with severe pathologies in mammals, including cancer (4-6). Signaling is initiated at the cell surface where the secreted, lipid-modified Wnt glycoprotein interacts with the extracellular N-terminal cysteine-rich domain (CRD) of the frizzled (FZD) receptor (7). This high-affinity interaction occurs at two distinct contact sites, one comprising a protein-fatty acyl interface and another a protein-protein interaction interface (8-10). Sequence analysis revealed that there are 10 human frizzled genes grouped into four clusters (SI Appendix, Fig. S1A).Among their multiple roles in physiology, FZD receptors have emerged as critical regulators of Wnt-dependent stem cell processes. For example, FZD7 is a critical component of the stem cell niche and was shown by genetic knockdown experiments to be essential for maintaining human embryonic and epithelial limbal stem cells in an undifferentiated state (11,12). Of the 10 mammalian frizzleds, FZDs 1, 2, and 7 are enriched at the base of mammalian adult intestinal crypts where the stem cells reside (13,14), and FZD7 is required for stem cell-mediated regeneration of the intestinal epithelium (15). Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors (16)(17)(18)(19)...

Non-acylated Wnts Can Promote Signaling

“…It is clear that the hydrophobic channel on the FZD CRD has a strong preference for binding fatty acids or other lipid-like molecules. Indeed, a recent crystal structure of the hFZD7 CRD showed the hydrophobic channel occupied by a fatty acid that originated in the cells used for protein expression – which do not overexpress a Wnt (Nile et al, 2017). In this study and another (DeBruine et al, 2017), it was further shown that binding of free fatty acids induces FZD-CRD oligomerization.…”

“…This structure confirmed that a conserved serine (S187 in xWnt8) is the only acylation site, and suggested that the S187-linked palmitoleoyl moiety plays a crucial role in FZD binding by occupying a hydrophobic channel on the CRD. This hydrophobic channel also binds free fatty acids in a manner thought to promote FZD oligomerization (DeBruine et al, 2017; Nile et al, 2017). Since all Wnts except WntD are predicted to be acylated at this conserved serine (Nile and Hannoush, 2016; Takada et al, 2006), it is thought that Wnts all engage and activate FZDs through such acylation-dependent interactions (Figure 1A).…”

Functional dissection of the N-terminal extracellular domains of Frizzled 6 reveals their roles for receptor localization and Dishevelled recruitment

“…The importance of the linker cysteines for receptor function is further underlined by previous reports identifying FZD 4 familial exudative vitreoretinopathy (FEVR) mutations in Cys-181 and Cys-204 of human FZD 4 (57)(58)(59). These C181R, C204R, C204Y mutations (corresponding to Cys-161 and Cys-185 in human FZD 6 , respectively) manifested with a trafficking phenotype and poor receptor surface expression (57,58).…”

“…FZDs, the focus of this study, bind WNT proteins with their extracellular cysteine-rich domain (CRD) to initiate a complex network of WNT signaling pathways (2). The CRD is seen as the orthosteric binding site for WNTs and is functionally implicated in receptor dimerization and signal initiation (3)(4)(5)(6). Structurally, the CRD is connected to the seven transmembrane (7TM) core of the receptor through a linker domain of variable length and low degree of conservation among the representatives of class F (5).…”

Mechanisms of intercellular Wnt transport

“…Furthermore, recent observations from the crystal structure of the Fzd7 CRD bound to a fatty acid has revealed that the palmitoleic lipid adduct binds to a U-shaped lipidbinding cavity of the Fzd7 dimer. Fzd5 and Fzd8 have similar architectures, including a dimeric arrangement of the CRD, suggesting a common model for how Wnt binds Fzd receptors via the fatty acid modification (Nile et al, 2017).…”