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
-
- 14,285
- views
-
- 2,095
- downloads
-
- 91
- 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
-
- Chromosomes and Gene Expression
- Structural Biology and Molecular Biophysics
Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The SARS-CoV-2 main protease (Mpro or Nsp5) is critical for production of viral proteins during infection and, like many viral proteases, also targets host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 is recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes cellular protein synthesis and redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. Evolutionary analysis shows the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. TRMT1 proteolysis results in reduced tRNA binding and elimination of tRNA methyltransferase activity. We also determined the structure of an Mpro-TRMT1 peptide complex that shows how TRMT1 engages the Mpro active site in an uncommon substrate binding conformation. Finally, enzymology and molecular dynamics simulations indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis following substrate binding. Together, these data provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.
