Structure of human Frizzled5 by fiducial-assisted cryo-EM supports a heterodimeric mechanism of canonical Wnt signaling

  1. Naotaka Tsutsumi
  2. Somnath Mukherjee
  3. Deepa Waghray
  4. Claudia Y Janda
  5. Kevin M Jude
  6. Yi Miao
  7. John S Burg
  8. Nanda Gowtham Aduri
  9. Anthony A Kossiakoff
  10. Cornelius Gati  Is a corresponding author
  11. K Christopher Garcia  Is a corresponding author
  1. Stanford University School of Medicine, United States
  2. The University of Chicago, United States
  3. Princess Máxima Center for Pediatric Oncology, Netherlands
  4. SLAC National Accelerator Laboratory, United States
  5. 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.

The following data sets were generated

Article and author information

Author details

  1. Naotaka Tsutsumi

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3617-7145
  2. Somnath Mukherjee

    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Deepa Waghray

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Claudia Y Janda

    Research, Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3307-2559
  5. Kevin M Jude

    Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3675-5136
  6. Yi Miao

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0738-6041
  7. John S Burg

    Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Nanda Gowtham Aduri

    Bioscience Division, SLAC National Accelerator Laboratory, Menlo Park, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Anthony A Kossiakoff

    Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Cornelius Gati

    Bioscience Division, SLAC National Accelerator Laboratory, Menlo Park, United States
    For correspondence
    cgati@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
  11. K Christopher Garcia

    Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, United States
    For correspondence
    kcgarcia@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9273-0278

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

  1. Yi Arial Zeng, Chinese Academy of Sciences, China

Publication history

  1. Received: May 1, 2020
  2. Accepted: August 6, 2020
  3. Accepted Manuscript published: August 7, 2020 (version 1)
  4. 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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  1. Naotaka Tsutsumi
  2. Somnath Mukherjee
  3. Deepa Waghray
  4. Claudia Y Janda
  5. Kevin M Jude
  6. Yi Miao
  7. John S Burg
  8. Nanda Gowtham Aduri
  9. Anthony A Kossiakoff
  10. Cornelius Gati
  11. K Christopher Garcia
(2020)
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

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