TY - JOUR
T1 - Structure of human frizzled5 by fiducial-assisted cryo-em supports a heterodimeric mechanism of canonical wnt signaling
AU - Tsutsumi, Naotaka
AU - Mukherjee, Somnath
AU - Waghray, Deepa
AU - Janda, Claudia Y.
AU - Jude, Kevin M.
AU - Miao, Yi
AU - Burg, John S.
AU - Aduri, Nanda Gowtham
AU - Kossiakoff, Anthony A.
AU - Gati, Cornelius
AU - Garcia, K. Christopher
N1 - Funding Information:
This research was supported by NIH grants 1R01DK115728 to KCG, R01GM117372 and P50GM082545 to AAK, and by the Department of Energy, Laboratory Directed Research and Development program at SLAC National Accelerator Laboratory, under contract DE-AC02-76SF00515, to CG. KCG is an investigator of the Howard Hughes Medical Institute and the Younger Family Chair, and is supported by the Ludwig Institute. NT was supported in part by the Long-Term Fellowships from the Human Science Frontier Program Organization (LT000011/2016 L).
Funding Information:
The cryo-EM data were collected at the Cryo-EM facility at the HHMI Janelia Research Campus; we thank Drs. Zhiheng Yu and Doreen Matthies for their generous supports in microscope operation and data collection. Preliminary cryo-EM screening is performed at the Cryo-EM Facility at the Department of Structural Biology, Stanford University and the Stanford-SLAC Cryo-EM facility; We thank Drs. Dong-Hua Chen and Elizabeth Montabana for their kind supports in operating the facili-ties and user training. Most of data were processed on the SLAC cluster; We thank Dr. Yee-Ting Li for support with computing resources. Dr. Satchal Erramilli assisted in generation of the anti-BRIL Fab. We thank Andrew Velasco for assistance, Caleb Glassman for scientific discussion. This research was supported by NIH grants 1R01DK115728 to KCG, R01GM117372 and P50GM082545 to AAK, and by the Department of Energy, Laboratory Directed Research and Development program at SLAC National Accelerator Laboratory, under contract DE-AC02-76SF00515, to CG. KCG is an investigator of the Howard Hughes Medical Institute and the Younger Family Chair, and is supported by the Ludwig Institute. NT was supported in part by the Long-Term Fellowships from the Human Science Frontier Program Organization (LT000011/2016 L).
Publisher Copyright:
© Tsutsumi et al.
PY - 2020/8
Y1 - 2020/8
N2 - 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/b-catenin signaling in response to Wnt3a versus a ‘surrogate agonist’ that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/b-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.
AB - 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/b-catenin signaling in response to Wnt3a versus a ‘surrogate agonist’ that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/b-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.
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U2 - 10.7554/ELIFE.58464
DO - 10.7554/ELIFE.58464
M3 - Article
C2 - 32762848
AN - SCOPUS:85089786899
SN - 2050-084X
VL - 9
SP - 1
EP - 20
JO - eLife
JF - eLife
M1 - e58464
ER -
