We screened a variety of vertebrate and invertebrate Wnts for expression and found that Xenopus
Wnt8 (XWnt8) expressed at high levels and could be purified as a complex with several Fz-CRD. XWnt8 is advantageous for structural studies as it has served as a model system to study Wnt/Fz interactions because it binds to and activates mammalian Fz (27
). A key enabling finding was that co-expression of XWnt8 with an Fc-Fz8CRD fusion allowed efficient affinity-based purification of XWnt8/Fz8-CRD complexes in the absence of detergent. In contrast, purification of Wnt alone requires detergent, suggesting that binding to the Fz-CRD shields the Wnt lipid from aqueous solvent (). The XWnt8/Fz-CRD complex eluted from gel filtration as two inter-converting oligomeric forms with MW ranging from ~50kDa to ~200kDa ().
Formation of the XWnt8 complex with mouse Fz8-CRD for structure determination
We crystallized the glycosylated XWnt8/Fz8-CRD complex in detergent-free buffers and obtained a native x-ray data set to a resolution of 3.25Å (Table S1
). Experimental phases were determined using isomorphous and anomalous scattering difference methodologies with crystals derived from material expressed in S2 cells supplemented with Selenomethionine (Table S1
). The experimental phases yielded an excellent electron density map in which XWnt8 could be traced, the Fz8-CRD located (), and the complex structure refined (Fig. S1
). The amino acid register of XWnt8 was confirmed using the Selenium sites as guides (), as well as the locations of disulfide bridges, N-linked glycans (Fig. S1
) and the lipid group.
The complex structure is a striking donut shape () in which XWnt8 appears to grasp the Fz8-CRD at two opposing sites using extended thumb and index fingers projecting from a central “palm” domain, to contact “site 1” and “site 2,” respectively, burying a total of ~2000Å2
of surface area. Neither the structure of XWnt8, nor the manner of Fz binding, bears a clear resemblance to known protein folds or complexes, respectively. XWnt8 comprises an N-terminal α-helical domain (NTD) from residues ~1–250 (helices A though F) that contains the lipid-modified thumb, and a C-terminal cysteine-rich region (CTD) from residues 261–338. Each domain forms a distinct interaction with the Fz8-CRD, whose conformation is essentially unchanged compared to the unliganded structure (Fig. S2A
), leaving a large hole in the center of the complex (, S2B
). The XWnt8 NTD is comprised of a seven α-helical bundle palm, containing two large inter-helical loop insertions that are stabilized by 4 disulfide bonds (). The principal feature of the CTD is a long 40 amino acid β-strand hairpin that is also stabilized by an extensive network of disulfide bonds. The distinct structural sub-domains associate through a small interaction patch between the AB-loop of the NTD and a small helix (helix F) in the CTD. There is clear electron density for high-mannose glycan additions at two of the three Asparagine-linked glycosylation sites on XWnt8, Asn104 and Asn263 (, S1b
Overall structure of XWnt8 in complex with Fz8-CRD
The functional role for lipid modification of Wnts is unknown, but has been shown to be necessary for full biological activity (18
). The structure shows XWnt8 lipidation directly involved in Fz8-CRD binding in binding site 1 (, ). A 15Å long tube of continuous electron density is connected to the hydroxyl group of Ser-187 (), which is located at the tip of the thumb projecting from the XWnt8 NTD. The length of the electron density corresponds to a 14-carbon lipid chain. The lipid dominates the contact interface, burying approximately 580Å2
of total surface area (330Å2
from the lipid, 250Å2
from the CRD), contacting 11 Fz8 residues, and completely traversing the cleft on the Fz8-CRD surface (). The lipid electron density is consistent with a 16-carbon palmitoleic acid (or derivative thereof) modification to XWnt8 where the terminal two carbons of the acyl chain have exited the CRD groove and do not show ordered electron density. Wnt acylation has been reported to be either an unsaturated palmitic acid, or a monounsaturated palmitoleic acid (23
). We could not unambiguously determine the chemical identity of the lipid on XWnt8 using mass spectrometry (). However, based on identification of the lipid attached to the corresponding Ser209 of human Wnt3a as palmitoleic acid, we assigned the lipid attached to XWnt8 Ser187 as palmitoleic acid - but it is formally possible that the lipid is palmitic acid. Serine acylation in the complex structure also resolves uncertainty regarding the location of the lipid attachment sites on Wnts. Both conserved Serine (209) and Cysteine (77) residues have been reported as acylation sites on human Wnt3a, and it has been speculated that other Wnts are acylated at one or both of the corresponding positions (18
). In XWnt8, we find that Cys55, the Cys residue analogous to Cys77 in Wnt3a, is engaged in a disulfide bond that will be conserved across all Wnts (, S3
), and so cannot serve as a lipid addition site. Therefore, the conserved Ser (corresponding to Ser187 in XWnt8) appears to be the consensus acylation site.
Acylation of the XWnt8 thumb loop mediates site 1 binding to Fz8-CRD
The cleft on the Fz8-CRD surface that is traversed by the lipid is made up of helix A, helix D and the DE-loop (, Table S2
), and is lined with hydrophobic amino that form extensive van der Waals interactions with the lipid (, Table S2
). The high degree of conservation of apolar amino acids in the region of the CRD contacting the acyl group implies that the lipid-binding site is conserved in other Fz-CRD (, S4
). The conservative substitutions seen for these residues in other Fz-CRD could modulate lipid-binding affinity and impart a degree of Wnt specificity (). The driving force for lipid binding appears to be the hydrophobic effect combined with shape complementarity of the lipid-CRD interface, where the lipid and apolar Fz-CRD core residues are driven to associate by solvent exclusion. Although approximately 60% of the total accessible surface area (~530Å2
) of the lipid is buried when bound to the Fz8-CRD, one face and the distal 2–3 carbon atoms of the lipid are still exposed to solvent. These exposed regions, ~200Å2
of hydrophobic surface, would be highly energetically unfavorable in aqueous solvent and may still require shielding.
While the site 1 interaction appears to a large degree mediated by the lipid on Wnt, thumb loop amino acids (residues 181–188) form protein-protein contacts with the Fz8-CRD that account for an additional ~600Å2
of buried surface area (). At the extreme tip of the thumb loop (residues 186–188) several main chain van der Waals contacts are formed with the Fz8-CRD that would have limited capacity to contribute to ligand specificity (). At the base of the thumb loop, Wnt Lys182 forms a salt bridge with the Fz8 Glu64 and a hydrogen bond with Fz Asn58. Lys or Arg are conserved at this corresponding position in all Wnts, and Glu or Asp are conserved at the Glu64 position in 8 of 10 mammalian Fz-CRD (Table S2
, Figs. S3 & S4
). However, the substitution of Thr and Ile in Fz3 and Fz6, respectively, raises the possibility of some degree of ligand specificity modulated through this interaction. We surmise that the principal driving force for the site 1 binding is the lipid-in-groove contact, with the residues at the base of the thumb contributing secondarily.
The highly exposed structural disposition of the lipid attachment site has several important implications. First, it suggests that lipid attachment may not be integral to the tertiary structural stability of the folded Wnt molecule. Clearly, acylation is necessary for proper secretion of Wnts, and our complex structure also reveals its centrality in Fz binding. But it should be possible to create viable Wnt protein therapeutics by genetically engineering “lipid-free” water-soluble Wnts through affinity maturation of Fz-contacting residues at the tip of the Wnt thumb. Second, the highly exposed position of the lipid suggests it would require sequestration from aqueous solvent during expression and transport, such as with carrier proteins (24
). In Wnt’s role as a morphogen, it has been suggested that Wnts may use acylation to partition into the cell membrane in order to increase local concentrations and restrict availability to specific target tissue (18
). The XWnt8 structure supports this idea in that the lipid is accessible (), ideally positioned for anchoring Wnt to the plasma membrane.
The site 2 interaction is on the opposite side of the Fz8-CRD from site 1 (), and is comprised of residues between the Cys315-Cys325 disulfide at tip of the XWnt8 CTD index finger, engaging in hydrophobic contacts within a depression between inter-helical loops on the CRD (, Table S2
). The site 2 interface buries a total of ~840Å2
XWnt8) and despite the “knob-in-hole” binding mode (), exhibits poor overall shape complementarity (Sc = 0.48). The XWnt8 index finger presenting the site 2 residues is a long, twisted β-strand, rigidified by a ladder of disulfide bonds, and spans from Gly299 to the C-terminal Cys338 (). In site 2, the underside of the finger loop positions hydrophobic residues Cys315, Phe317, Trp319, an unusual tandem Cys320-Cys321 disulfide bond, and Val323 to form the major van der Waals interactions with main chain and apolar residues on the Fz8-CRD (). The XWnt8 Trp319 side chain at the tip of the finger loop occupies a pocket on the Fz8-CRD surface and engages primarily the main-chain of Fz8-CRD residues 150–152, and the side chain of conserved Phe86. The XWnt8 site 2 contact residues are invariant in all Wnts (Fig. S3
). In the Fz8-CRD Tyr48 and Cys148, are conserved residues that form van der Waals interactions with XWnt8 (Fig. S4
). As for site 1, Wnt and Fz contact residues are conserved apolar amino acids (, S3
). Importantly, several Fz8-CRD contacts are substituted in other Fz-CRDs and thus could contribute to Wnt sub-type preferences. For example, Met149 at the center of site 2 is conserved in 5 of 10 mammalian Fz-CRD, but is substituted to Val, Glu or Asp in Fz1, 2, 3, 6 and 7.
A conserved Wnt/Fz recognition mode in the site 2 interface
Given the technical difficulties of expressing recombinant Wnts, there is a dearth of structure-function data, or biochemical measurements between Wnt and Fz. Here, guided by the structure of the complex, we engineered a biochemically tractable version of XWnt8 in order to determine three previously unknown interaction parameters: 1- the degree to which XWnt8 site 1 versus site 2 binding determines Fz specificity, 2- if the site 1 and 2 interactions can occur independently, or whether both sites are reliant on simultaneous engagement to achieve productive binding, and 3- measurement of an accurate binding affinity of site 2 alone. For the first experiment, we displayed a water-soluble, C-terminal 90 amino acid sub-domain of XWnt8, containing the site 2 binding index finger (which we term “mini-Wnt”) on yeast. We tested Fz1, 2, 4, 5, 7 and 8 CRD for binding and clearly observed that mini-XWnt8 was stained by FACS with several Fz-CRD (Fz4, Fz5 and Fz8) that were presented as fluorescent tetramers by forming complexes with Streptavidin-PE (). Interestingly, we see stronger binding to Fz8 (~50% staining) and Fz5 (~30% staining) than Fz4 (~5% staining), as measured by FACS staining intensity. The soluble mini-Wnt binding to Fz8, Fz5, and Fz4 is in accord with full-length XWnt8 previously determined in a cell binding assay (27
). The concordance of binding specificity between mini-Wnt and full-length XWnt8 demonstrates that Fz discrimination is mediated primarily, although we cannot say exclusively, by XWnt8 site 2, and also that the site 2 contact can occur independently of site 1. The lipid on the Serine in the site 1 contact by full-length Wnt, which is clearly necessary for full Wnt activity, may also be important for affinity enhancement and tissue localization. In order to determine an accurate affinity of site 2, we produced soluble mini-Wnt in a recombinant form expressed from insect cells, and measured a KD
of ~2.4uM for Fz8-CRD, and ~3.5uM for Fz5-CRD using surface plasmon resonance (). From these studies we conclude that site 2 alone represents a moderate affinity interaction site that binds to three different Fz, but also has differences in binding affinity for different Fz, demonstrating that site 2 is not entirely degenerate. While the mini-Wnt affinity difference between Fz5 and Fz8 is small, it is consistent with the yeast display rank order of preference, and the relative difference may be amplified in the context of both sites in full-length Wnt to a degree that could functionally discriminate Fz receptors. We surmise that site 1 and site 2 combine to manifest as high affinity for Wnts through two-point attachment. Collectively, the biological relevance of the site 1 and site 2 interactions we see in the crystal structure are supported three lines of evidence: 1- the concentration of amino acid conservation patterns in both XWnt8 and the Fz8-CRD interfaces, 2- the direct involvement of the lipid group in binding given functional data showing the necessity of Wnt3a Serine palmitoylation for activity, and 3- both current () structure-function data on XWnt8 (), and prior mutational data mapping the Wnt binding site on Fz-CRD (26
“Mini-Wnt” discriminates between different Fz-CRD and engages site 2 independently of site 1