Structural Studies of Wnts and Identification of an LRP6 Binding Site

Abstract

SUMMARY Wnts are secreted growth factors that have critical roles in cell fate determination and stem cell renewal. The Wnt/β-catenin pathway is initiated by binding of a Wnt protein to a Frizzled (Fzd) receptor and a co-receptor, LDL receptor-related protein 5 or 6 (LRP5/6). We report the 2.1Å resolution crystal structure of a Drosophila WntD fragment encompassing the N-terminal domain and the linker that connects it to the C-terminal domain. Differences in the structures of WntD and Xenopus Wnt8, including the positions of a receptor-binding β-hairpin and a large solvent-filled cavity in the helical core, indicate conformational plasticity in the N-terminal domain that may be important for Wnt-Frizzled specificity. Structure-based mutational analysis of mouse Wnt3a shows that the linker between the N- and C-terminal domains is required for LRP6 binding. These findings provide important insights into Wnt function and evolution.

Disulfide Bond Requirements for Active Wnt Ligands

“…3), suggesting further that c3 (Cys-77) is unlikely to be palmitoylated but rather forms a disulfide bond with c4 as seen in Xwnt8 and WntD (18,21). This notion is consistent with other recent experimental evidence (15,19).…”

“…Alternatively, the oxidized oligomer formation of these Wnt3a mutants may occur gradually after secretion, permitting signaling from the transient monomeric form before oxidized oligomers accumulate. Consistent with either of these possibilities, the areas surrounding cysteines c3-c8 neither interact with the FZD CRD in the Xwnt8 co-crystal nor overlap with the predicted or experimentally mapped LRP6 binding regions (18,20,21). In the index finger, mutation at either of the more proximal cysteines (c18 or c23) partially reduced activity, whereas the more distal pair (c19 and c22) displayed a severe reduction of activity (Fig.…”

“…Interestingly, a shorter Xwnt8 truncation lacking an additional 30 amino acids from the linker domain did not exhibit the effective dominant negative property (28), possibly due to truncating into the prospective LRP6 binding region. A similarly truncated and dominant negative Wnt3a mutant was recently reported (21). Further Analysis of the Wnt3a Thumb Region-Having validated experimentally the key aspect (the vicinal disulfide bond at the tip) of the Wnt3a index finger, we further examined a cardinal feature of the thumb, the serine residue (Ser-209) that is palmitoleoylated.…”

“…Drosophila WntD (Wnt inhibitor of Dorsal) is an unusual Wnt protein in that it harbors a glutamine instead of a serine at the thumb tip (31,32). Unlike all of the Wnt proteins that have been examined thus far, WntD is secreted efficiently independent of Porcupine and Wntless functions (33) despite the fact that it has a similar structural fold as Xwnt8 (21). Locations of the ␤ sheets and disulfide bonds in hairpin 2 in the WntD thumb are identical to those in Xwnt8, although WntD contains three additional amino acids in the thumb (Fig.…”

“…A crystallized interface between a Wnt ligand and the LRP6 co-receptor is yet to be reported; however, a constellation of conserved residues in the Wnt palm region probably contribute to LRP6 binding (18,20). Indeed a series of positively charged residues in the Wnt3a palm region were recently shown to be important for LRP6 interaction (21).…”