Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity.

Sumit Kumar,Michael Boutros,Trushar R Patel,Jörg Stetefeld,Herbert Steinbeisser,Dietmar Gradl,Thomas Holstein,Benjamin Trageser,Julia Christina Gross,Suat Özbek,Karolin Rahm,Mihaela Žigman

doi:10.1186/1741-7007-12-44

Abstract

BackgroundWnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed.ResultsHere, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner.ConclusionsWe propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.

Highlights

Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans
Mutational analysis of in vitro Wnt3a activity To predict critical sites in mouse Wnt3a required for Fz binding, we generated a structural model of the mouse Wnt3a-Fz8-CRD complex adopting the recently published X-ray structure (Figure 1A,E)
As demonstrated by Janda et al, the Wnt molecule contacts the Fz-CRD at two distinct binding sites within the N-terminal domain (NTD) and C-terminal domain (CTD) designated as sites 1 and 2, respectively (Figure 1A)

Summary

Introduction

Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The crystal structure of the Wnt8-Frizzled8-cysteine-richdomain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed. Wnts are key mediators of developmental processes throughout the metazoan kingdom [1,2,3]. Janda et al [9] presented the crystal structure of the Xenopus Wnt in complex with mouse Fz8CRD. Wnt was shown to have a two-domain structure in which the alpha-helical N-terminal domain (NTD) formed a contact with Fz via the palmitoic acid lipid group (site 1) whereas the conserved region in the C-terminal domain (CTD) formed a strong hydrophobic contact with a groove in the Fz8-CRD (site 2). Whether and how site 1, 2, and 3 residues impact on biological Wnt function under relevant physiological conditions remained unknown

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