Wnt- Fz Structure & interactions

Figure 1
Bound Wnt in Frizzled receptor. Shown in cyan is the Wnt
protein, and shown in green is the Frizzled receptor.
Shown as spheres are organic molecules. Brick red is NAG,
Blue is BMA and yellow is MAN.

Wnt and the frizzled (Fz) receptor from a characteristic doughnut shaped complex (Figure 1). The contacts between the two proteins are mediated through two main binding sites.
Purification and crystallisation of the complex requires no detergent as Frizzled largely protects the highly hydrophobic lipid attachment on the Wnt. The lipid attachment on Wnt is necessary for biological function and is thought to localise the protein to the membrane, chaperones would be needed to protect the lipid group in solution when secreted.

Binding site 1

binding site 1, preliminary takes advantage of the palmitoleic lipid group attached to the tip of the Wnt Thumb at Serine 187, whereby the Fz has a hydrophobic groove with strongly conserved amino acid residues either, fully or chemically. This site 1 interaction is driven largely by the hydrophobic effect and involves solvent exclusion during binding, as well as shape complementarity.

Figure 2
This image, created on pymol, shows the Frizzled CRD in
cartoon form, with an outline of the surface (at 0.5 transparency).
Shown is blue is the hydrophobic surface of the groove,
which interacts with the Wnt- lipid attachment.
Note: glycan attachments have been omitted.

Figure 3
Frizzled CRD depicted in cartoon form,
Highlighted in blue are the regions interacting with
the Wnt-Lipid attachment.

The contact between the Wnt-Palmitoleic lipid group and the Fz is mediated by 10 hydrophobic amino acid residues (Q71, F72, P74, L75, I78, L121, M122, Y125, F127 and P130) found on helix A, D and the inter helical loop between helix D and E (see figure 3), forming extensive van der Waals interactions between the lipid and the Frizzled receptor ( to see conserved residues please refer to figure 2 here ). This binding interaction accounts for a 580 Armstrong squared binding surface (330 from the surface of the lipid and a  further 250 Armstrong's squared of the Fz surface).

The interaction between these amino acid residues and the lipid can be seen in figure 4.

Figure 4
Pymol image showing ribbon form of the Frizzled
CRD as well as the surface of the protein set at
0.5 transparency. Seen in red is the palmitoleic
acid attachment of the Wnt, in blue the
hydrophobic amino acids interacting with the
lipid are highlighted.

While most of the highly hydrophobic lipid is buried in the groove of the receptor, the last 2-3 carbons and one face of the lipid are outside of this groove, hence exposing about 200 Armstrong's squared of the lipid surface to solvent. This is highly entropically unfavourable hence probably requires further shielding to minimise the entropic debt.

There is also a secondary interaction between Wnt and Fz at the base of the thumb loop of Wnt. The thumb loop Wnt amino acids 181-188 form protein/ protein interactions with the Fz receptor. This interaction accounts for the burial of a further 600 Armstrong's squared. At the base of the thumb loop there is a strictly conserved lysine 182, which forms a slat bridge with the 8/10 conserved amino acid residue; Glutamic acid 64 of the Frizzled receptor as well as a hydrogen bond with the strictly conserved amino acid residue asparagine 58 of Frizzled CRD. (see figure 6)

At the extreme tip of the thumb loop there are some main chain amino acid van der Waals interactions with Frizzled but these do not contribute to binding specificity rather they just reinforce the binding. 

Figure 6
In white we see part of the Frizzled receptor surface, where the dark blue represents
the areas in contact with the lipid (not shown). In cyan we see Wnt. The image shows
the conserved amino acids Glu64 forming a salt bridge with Lys 182 (yellow dotted line)
and the conserved amino acid Asn 58 which forms a hydrogen bond (red dotted line)
with lys 182 of Wnt.

Figure 7
Image created on pymol showing primary and secondary interactions of binding site 1.
Shown in Cyan is Wnt, highlighting with sticks Lys 182 which forms a salt bridge
and a hydrogen bond with Fz amino acids (described above) Shown in red is the
palmitoleic lipid attachment of Wnt at Ser 187.

Figure 8
This pymol image shows all the site 1 interactions between Wnt and the Frizzled receptor. Including
the Hydrophobic interaction between the palmitoleic acid and hydrophobic amino acid residues
in the Frizzled CRD groove. Furthermore the extreme tip of the Wnt thumb is highlighted where
a network of van der Waals interactions are established. Note that not the whole Wnt structure
is portrayed here only the relevant regions with respect to interactions in binding site 1.

Binding site 2

Binding site two is located on the opposite side of the Fz-CRD with respect to binding site 1 and buries about 840 Armstrong's squared of surface area, 400 of which is on the Fz- CRD and 440 oh which is on the Wnt. This binding site has poor overall shape complementarity. Despite this it seems that Fz discrimination between different strains is largely mediated by site 2 interactions. This is due to the fact that binding site 1 is dominated by the lipid interaction which is non specific for Wnt/Fz types. It is also note worthy that site 2 interactions can occur independently from site 1 interactions. This was determined using "mini Wnts" whereby the Wnt is modified and truncated so that it only contains certain soluble structural regions.

Different Wnt can bind to different Fz to some degree however they do show a quantitative discrimination between different wnt/fz pairs showing that binding site 2 is not broadly degenerate.

Binding site 2 consists of amino acids cys315 - cys325 on the Wnt CDT index finger tip and a groove comprised of helical loops on the Fz-CRD containing hydrophobic residues. The under side of the finger loop of the Wnt forms the main interactions with Fz. This area consists of hydrophobic residues Cys 315, Phe 317, Trp 319, a rare tandem of Cys 320 and Cys 321 forming a disulphide bond and Val 323 forming major main chain van der Waals interactions.

At the tip of XWnt8 finger loop a Trp139 side chain engages with main chain of Fz-CRD residues 150-152 and side chain of the conserved residue Phe 86 forming a pocket.

Further conserved residues in the Fz CRD in binding site 2 consist of Tyr 48 and cys 148 these also form van der waals interactions with Wnt.

While much is known now of the interactions between Wnt and Frizzled, the activation mechanism of Frizzled is thus far un-clear. This is partly due to the fact that the tertiary structure of Fz-CRD remains nearly unchanged upon binding. 

figure 9Pymol image shows the Frizzled CRD in green and the
index finger loop of Wnt. Shown in purple are the amino
acids Cys 315- Cys 325 which partake in the binding.
Shown in blue is the underside of the index finger loop
which form the main van der Waals interactions. In addition
Trp 139 is fully shown as it sinks into the Fz pocket.
Furthermore on the Fz-CRD highlighted in Orange is the
Conserved Phe 86 and in red the conserved Tyr 48 and Cys 148.

Figure 10
Pymol image showing site 2 interactions. Seen in green is the
Fz-CRD and in cyan the Wnt molecule. Structures are shown in
ribbon form with a 0.5 transparency surface.

Oligomerisation and Crystal Contacts
While there is some evidence that would support an oligomerisation theory there is no symmetric dimmer in the crystal, however there is a third contact, termed the 'pseudo site 3' in the crystal. This third contact is the largest on in the crystal an buries the remaining 200 Armstrong's squared of surface left exposed on the lipid. This contact mediates the formation of an asymmetric Wnt-Fz dimer. The interface consists of 1 Wnt molecule binding to the lipid exposed surface of a Wnt-Fz-CRD complex. The asymmetry of this site in the crystal produces self associating repeating units of complexes. The physiological relevance of this site is thus far unknown however it is speculated that pseudo site 3 provides a basis for both ligand induced receptor clustering as well as the dishevelled singalosome assembly.

All Information on this page is taken from
Janda, C.Y. et al., 2012. Structural basis of Wnt recognition by Frizzled. Science (New York, N.Y.), 337(6090), pp.59–64

Structural basis of Wnt recognition by Frizzled

Structural basis of Wnt recognition by Frizzled