Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling
- Stanford University School of Medicine, United States
- The University of Chicago, United States
- Princess Máxima Center for Pediatric Oncology, Netherlands
- SLAC National Accelerator Laboratory, United States
- Howard Hughes Medical Institute, Stanford University School of Medicine, United States
Abstract
Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/β-catenin signaling in response to Wnt3a versus a 'surrogate agonist' that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/β-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine.
Data availability
The cryo-EM map has been deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-21927 and the model coordinate has been deposited in the Protein Data Bank (PDB) under accession number 6WW2.All cell-based assay data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2 (Figure 2 - Figure supplement 1) and Figure 4 in excel format.
Article and author information
Author details
K Christopher Garcia
Funding
National Institutes of Health (1R01DK115728)
- K Christopher Garcia
Howard Hughes Medical Institute
- K Christopher Garcia
Ludwig Institute for Cancer Research
- K Christopher Garcia
National Institutes of Health (R01GM117372)
- Anthony A Kossiakoff
National Institutes of Health (P50GM082545)
- Anthony A Kossiakoff
U.S. Department of Energy (DE-AC02-76SF00515)
- Cornelius Gati
Human Frontier Science Program (LT000011/2016-L)
- Naotaka Tsutsumi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Yi Arial Zeng, Chinese Academy of Sciences, China
Version history
- Received: May 1, 2020
- Accepted: August 6, 2020
- Accepted Manuscript published: August 7, 2020 (version 1)
- Version of Record published: August 21, 2020 (version 2)
Copyright
© 2020, Tsutsumi et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling
eLife 9:e58464.
https://doi.org/10.7554/eLife.58464
Further reading
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- Cell Biology
- Structural Biology and Molecular Biophysics
Advanced cryo-EM approaches reveal surprising insights into the molecular structure that allows nascent proteins to be inserted into the membrane of the endoplasmic reticulum.
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- Developmental Biology
- Structural Biology and Molecular Biophysics
The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, Brachydactyly B and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.
