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.
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.
Metrics
-
- 13,550
- views
-
- 2,018
- downloads
-
- 80
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.
-
- Structural Biology and Molecular Biophysics
Complementary structural biology approaches reveal how an agonist and a covalent inhibitor simultaneously bind to a nuclear receptor.
