XWnt8 in complex with the cysteine-rich domain of Frizzled 8

Emma Klug '18 and Jess Khrakovsky '18

Contents:

I. Introduction

Wnt proteins are critical, highly conserved mediators of development in both vertebrates and invertebrates. They are secreted morphogens that establish gradients of transcription factor expression in progenitor cells, ultimately affecting cell proliferation, differentiation, and migration. Wnt, in conjunction with other morphogens, are responsible for the patterning of the neural tube early in embryonic development. They are also reused later in development as axon guidance molecules, and aid in synaptic formation. Wnt signaling is not only required in development, but also necessary for adult tissue maintenance.

Wnt binds to the G protein-coupled receptor Frizzled. In the canonical pathway, the interaction of Wnt with Frizzled is the first step in stimulating the transcription of Wnt target genes via the transcription factor Beta-Catenin. Functional Wnt/Frizzled signaling is required for proper development, and dysregulation of the system is implicated in many human diseases and cancers, including abnormalities in bone density, tooth development, and the retina. Here we dive into this crucial interaction via exploration of the structural basis of Xenopus Wnt8 (XWnt8) in complex with the mouse Frizzled-8 (Fz8) cysteine-rich domain (CRD).

Wnt signaling pathway:

 

II. Complex structure

Color Scheme:

Polypeptide Chain:

The Xenopus Wnt (XWnt8) is shown in complex with the cysteine-rich domain (CRD) of the mouse Frizzled-8 (Fz8). Frizzled receptors are found exclusively at the plasma membrane on the surface of Wnt-responsive cells. The extracellular CRD at the amino terminus of Frizzled binds Wnts with high affinity. XWnt8 and Fz8-CRD in complex form a distinct donut shape. XWnt8 makes contact with Fz8 at two distinct on opposite sides of the CRD.
 

III. Wnt Structure

Wnt proteins play a large, diverse role across species, thus an understanding of their structure is crucial. Xenopus Wnt 8 (XWnt8) has the appearance of a hand with a thumb and forefinger protruding from a thicker palm region, reaching out to pinch the cysteine rich binding domain (CRD) of its Frizzled (Fz) receptor. It is comprised of an N-terminal domain containing the thumb and palm regions, and a C-terminal domain containing the forefinger.

The palm of the NTD contains 2 large interhelical loop insertions stabilized by four disulfide bonds. The CTD is also stabilized by an extensive network of disulfide bonds. .

Wnt is subject to post-translational including palmitoylation and glycosylation. Both modifications are necessary for proper secretion, activity, and engagement of the protein with the Fz receptor. N-linked glycans are visible at and Patmitoylation at is especially important, as it forms the basis for interaction with Fz8 at binding site one.

Post-translational serine palmitoylation:

IV. Frizzled Receptor and the Frizzled Cysteine Rich Domain


Frizzled receptors are 7-pass transmembrane proteins containing an extracellular cysteine-rich domain (CRD) at the amino terminus that binds Wnt proteins with high affinity. The Fz8-CRD possesses 10 conserved within a domain of 120-125 amino acids. The CRD is predominantly alpha helical, with all cysteines forming disulphide bonds. The residues engaging in Wnt/Fz contact are highly conserved in Wnt proteins, however several contact residues found in the Fz8-CRD are substituted in other Frizzled-CRDs. For example, , found in binding site 2, is conserved in 5 of 10 mammalian Fz-CRDs, but can be substituted with valine, glutamic acid, or aspartic acid in other Frizzled receptors. This most likely provides the specificity of Frizzled receptors for subtypes of Wnt proteins. 

V. Binding Site I

The palmitoleic acid lipid group attached to the XWnt8 thumb directly engages a on Fz-CRD. This groove is lined with amino acids that form extensive van der Waals interactions with the lipid. In addition to the hydrophobic effect, Wnt Lys182 forms a with Fz8 Glu64 and a with Fz8 Asn58. Furthermore, thumb loop form protein-protein contacts with the Frizzled CRD. The primary driving force for the site 1 contact is the palmitoleic acid-protein hydrophobic interaction, while protein-protein interactions involving residues at the base of the thumb and slight shape complementarity are secondary.


VI. Binding Site 2

The second Wnt/Fz binding site is located opposite from site 1. It is composed of between the Cys315- Cys325 disulfide bond at the tip of the XWnt8 index finger, engaging in hydrophobic interactions within a depression between of the Fz8-CRD. The index finger reaching out to site 2 is a long, spanning from Arg301 to the C-terminal Cys321. The finger positions Cys315, Phe317, Trp319, the unusual tandem Cys320-Cys321 disulfide bond, and Val323 to form significant van der Waals interactions with the apolar residues on the CRD. The XWnt8 Trp319 sidechain at the tip of the finger sits in a pocket of the CRD surface and with Frizzled residues 150 to 152. Other essential, conserved interactions occur between Fz8 Tyr48 and Cys148, forming van der Waals with XWnt8.

Overview of Wnt/Fz interactions:

 

VII. References
Bazan, J., Janda, C., & Garcia, K. (2012). Structural Architecture and Functional Evolution of Wnts. Developmental Cell, 23(2), 227-232.
Huang, H., & Klein, P. S. (2004, June 14). The Frizzled family: Receptors for multiple signal transduction pathways. Genome Biology, 5(7), 234.1-234.7.

Christie, W. (. (n.d.). Proteolipids. Retrieved December 10, 2016, from http://www.lipidhome.co.uk/lipids/simple/protlip/index.htm

Janda, C. Y., Waghray, D., Levin, A. M., Thomas, C., & Garcia, K. C. (2012). Structural Basis of Wnt Recognition by Frizzled. Science, 337(6090), 59-64.

Komekado, H., Yamamoto, H., Chiba, T., & Kikuchi, A. (2007). Glycosylation and palmitoylation of Wnt-3a are coupled to produce an active form of Wnt-3a. Genes to Cells, 12(4), 521-534.

Kurayoshi, M., Yamamoto, H., Izumi, S., & Kikuchi, A. (2007). Post-translational palmitoylation and glycosylation of Wnt-5a are necessary for its signalling. Biochemical Journal, 402(3), 515-523.

Nusse, R. (2005). Wnt signaling in disease and in development. Cell Research, 15(1), 28-32, 28-32.

Wnt signaling in C. elegans*. (n.d.). Retrieved December 15, 2016, from http://wormbook.org/chapters/www_wntsignaling.2/wntsignal.html

Christie, W. (. (n.d.). Proteolipids. Retrieved December 10, 2016, from http://www.lipidhome.co.uk/lipids/simple/protlip/index.htm

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