Pdb 5Uwg, Pdb 5Bpb Rmsd = 1.12 Å, 1.15 Å (245 Cα)

a b

dimer / dimer refinement 5UWG aligned to 5BPB tetramer refinement RMSD = 14.22 Å (2807 atoms) RMSD = 0.44 Å (232 Cα) c

PDB 5UWG, PDB 5BPB RMSD = 1.12 Å, 1.15 Å (245 Cα)

Figure S1. FZD4 CRD crystallized with Palmitoleic Acid as a tetrameric complex. (A) Refinements of crystal structure using either dimer or tetramer as base unit yield similar results. (B) Tetramer refinement (PDB 5UWG) is similar to symmetry observed in previously published

FZD4 CRD structure by Chang et al. (PDB 5BPB). (C) Dimer constituents in tetramer refinement are highly similar to those contained in Chang et al. structure. a

PDB 5BPB PDB 5CM4 PDB 5BPQ Not crystallized 1.12 Å (245 Cα) 0.96 Å (298 Cα) 0.58 Å (298 Cα) independently 1.15 Å (245 Cα) 1.08 Å (298 Cα) b

XWnt8

FZD4 CRD

FZD CRD 4 Top-down view Side view c

XWnt8

FZD4 CRD Tetramer SMO linker Top-down view SMO linker

Figure S2. FZD4 CRD tetramers are constructed from known FZD4 CRD complexes and may bind Wnt ligands.

(A) FZD4 CRD tetramers are constituted of four unique monomer-monomer orientations, three of which have been previously crystallized.

(B) Wnts can bind FZD4 tetramers and reveal a complex with very little steric clashing. Here, FZD4 CRDs were aligned to the FZD8-CRD/XWnt8 structure (1.00±0.04 Angstroms over 132 Cα atoms).

(C) The Smoothened CRD-to-TMD linker was modeled onto the FZD4 CRD tetramer (PDB 4L7I) shown in blue and orange. One linker significantly clashes with Wnt (inset), but it is unclear whether the longer, more flexible FZD4 linker may adopt different orientations. a L132/L121 FZD8 FZD4

F82/F72

F137/F127

L85/L75

F135/Y125

b c 16 1 2 15 4 3 14 1 5 6 Δ9 2 11 13 12 4 7 8 3 9 10 5 6 7 8 9 10 1112 13 14

Figure S3. Mode of fatty acid binding in XWnt8/FZD8 CRD structure (PDB 4F0A) and FZD4 CRD dimer structure (PDB 5UWG). (A) Comparison of FZD4 CRD in complex with palmitoleic acid (PDB 5UWG) and FZD8 CRD in complex with lipidated XWnt8 (PDB 4F0A). Hydrophobic residues which coordinate with palmitoleic acid are positioned similarly in both structures. Only a single FZD4CRD is shown. (B) Unsaturated palmitoleic acid (red) adopts a bent conformation in complex with FZD4 CRD dimers, while Wnt-modifying lipid group (cyan) appears linear when crystallized with only a single FZD8 CRD. (C) Palmitoleic acid crystallized with FZD4 (red) is offset 3 carbons compared to Wnt- modifying lipid (cyan), and also adopts a bent conformation due to a Δ9-desaturation. a b

XWnt8

Norrin–Norrin

FZD4 CRD FZD4 CRD FZD4 CRD

Figure S4. Frizzled palmitoleic-acid-bound dimers can bind classical Wnts but not Norrin.

(A) Structure of XWnt8 is modeled to FZD4 CRD dimers by alignment of FZD8 CRD (RMSD 1.03 Å over 132 Cα atoms, PDB 4F0A) revealing that this dimer structure is sterically compatible with Wnt ligand binding.

(B) Norrin homodimers bind two independent FZD4 CRDs at distant surfaces facing opposite directions, inconsistent with a lipid-bound FZD4 CRD structure. a CRD b

Linker ECL3

SMO

SMO FZD3 FZD4 FZD5 FZD7 FZD9 0% 100% hFZD1-10 sequence homology

Figure S5. Comparison of the CRD-TMD linkage of Smoothened with that of Frizzled family members. (A) Smoothened extracellular domain with transmembrane domains showing protection of the CRD (orange) sterol-binding groove by CRD-to-transmembrane linker (pink) and long extracellular loop 3 (blue). (B) Ratio of linker to extracellular loop 3 (ECL3) length as a measure of flexibility between the transmembrane domain and CRD. Data calculated using SwissProt domain assignments. Frizzled subfamily members have similar linker:ECL3 length ratios. (C) Homology models of representative Frizzled subfamily members compared to Smoothened, highlighting the unconserved ECL3 and long, flexible linker domains. Conservation analysis was performed between the ten human Frizzleds. Structural models were generated using I-TASSER. 10000 -Wnt5a, +FZD4, +ROR2

+Wnt5a, +FZD4, +ROR2 7500

5000

2500 Luciferase Signal Luciferase 0 AP1-Luc - + + + + + + PMA (ng/mL) - - 6 18 - 6 18

Figure S6. Additional controls for WNT5A/AP-1 luciferase reporter system

HEK293 cells transiently transfected with FZD4 and ROR2 were co-transfected with AP1- Luciferase reporter plasmid and WNT5A as indicated. PMA was added 24 hours after plating and readings were taken another 24 hours after PMA addition. Error bars = S.E.M.