xWnt8 Structure



This image shows the xWnt8 protein. Any hydrophobic residues
are coloured red. The absence of red residues on the surface
of the protein show that is is hydrophillic. (The lipid addition is
 coloured red.)

Previously, there have been no structural analysis of Wnts. The primary sequence contains no similarity to other protein folds, thus making it difficult to predict 3D structure. 

C. Janda et.al use Xenopus frog Wnt8 for study as it can activate mammalian Frizzled (Fz) receptors. 

Crystallization of Wnts would require use of detergent due to their hydrophobic lipid modification. However, the complex of xWnt8 with its frizzled receptor was successfully crystallized without detergent, allowing the 3D structures to be revealed. 

Figure 1. xWnt8 protein. D1 is highlighted in green
D2 is highlighted in blue.
PDB file: 4F0A (PyMOL 1.3.)

Methods used by C. Janda et.al 

Gel filtration - Isolated the xWnt8-Fz complex. 

X-ray data -  native x-ray data set to a resolution of 3.25Å.

xWnt8 Structure

The overall structure of the xWnt8 protein can be related to a hand, where the 'thumb' and 'finger' extensions (seen in figure 1) clasp the Fz receptor at binding sites 1 and 2.The protein is made up of two distinct domains, D1 and D2.

D1, the N-terminal domain (NTD) consists approximately of residues 1-250, whereas D2, the C-terminal domain, extends from residues 261-338. (C. Janda et.al, 2012)

Note: The red loop is not classed as either domain and acts as a linker between D1 and D2.

D1. N-terminal Saposin-like domain

D1, made up of the 'palm'  and 'thumb', contains the N-terminus region of the protein. Figure 2 shows that the 'palm' is made up of seven alpha helices with two loop extensions that are stabilized by four disulphide bonds. 

Figure 2. xWnt8 N-terminal 'D1' domain.
The 7 helices of the 'palm' are coloured in green. 
The two interhelical loops are coloured in yellow and purple. 
Disulphide bonds are highlighted in red, with their 
cysteine residues labelled. PyMOL file 4F0A. 

'Thumb'

The two extending loops including four beta-strands, make up the 'thumb' region. They in turn are supported by four of the 'palm' helices that they are attached too.

Wnts are palmolipidated on a residue at the tip of the thumb which has shown to be important for Wnt signalling, often Histidine. (Willert et al. 2003). 

Figure 3. A lipid modification extends from 
Ser187 on the tip of the D1 'thumb'.

Figure 4. D2 domain of xWnt8. 
The disulphide bonds are 
coloured in red. 

xWnt8 has its lipid modification on Ser-187, located at the tip of the second beta-hairpin. (see figure 3). The lipid is attached to the hydroxyl group of the serine via an ester bond.  The first beta-hairpin is less structured and party collapsed in comparison to the second. (Bazan,J. Fernando. et al. 2012) 

D1 contains a saposin-like fold that resembles the apo helical form of a saposin protein domain.

D2. C-terminal domain 'Cysteine-rich region'.

The D2 domain has a long beta-strand hairpin structure which is stabilized by many disulphide bonds, seen in figure 4. T
his structure is labelled as the 'finger' of the Wnt.

Glycosylation

The Wnt has two other major sugar modifications. These are high-mannose post-transcriptional additions.

At Asn-104 there are NAG-NAG-BMA-MAN

And at Asn-263 there is NAG-NAD-BMA-FUC. (Janda et al. 2012) See PBD = 4F0A.

Conserved residues

Figure 5

A patch of conserved residues on xWnt8, coloured pink, are possible binding sites for co-receptors such as Lrp5/6, Ryk and Ror2.

The residues labelled are 216-219, 249-252 and 256-259. Conservation determined using ClustalW (not shown). 

Janda, C.Y. et al., 2012. Structural basis of Wnt recognition by Frizzled. Science (New York, N.Y.), 337(6090), pp.59–64.

Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, et al. 2003. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423:448–52

Bazan, J. ., Janda, C. & Garcia, K. . (2012) Structural Architecture and Functional Evolution of Wnts. Developmental Cell. 23 (2), 227-232.