Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma
Abstract
When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localizationmore »
- Authors:
-
[1];
[1];
[1];
[2];
[1];
[1]
- University of California, San Diego, CA (United States)
- University of Michigan, Ann Arbor, MI (United States)
- Rockefeller University, New York, NY (United States)
- Publication Date:
- Research Org.:
- Univ. of Michigan, Ann Arbor, MI (United States); Univ. of California, San Diego, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division (CSGB); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)
- OSTI Identifier:
- 1903844
- Grant/Contract Number:
- AC02-05CH11231; P30 GM124169; S10OD018483; T32 CA009523; R01 GM34921; R35 GM130389; S10OD020011
- Resource Type:
- Accepted Manuscript
- Journal Name:
- PLoS Biology (Online)
- Additional Journal Information:
- Journal Name: PLoS Biology (Online); Journal Volume: 18; Journal Issue: 12; Journal ID: ISSN 1545-7885
- Publisher:
- Public Library of Science
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; electron cryo-microscopy; crystal structure; molecular dynamics; allosteric regulation; DNA structure; biochemical simulation; nucleotides; small-angle scattering
Citation Formats
Lu, Tsan-Wen, Aoto, Phillip C., Weng, Jui-Hung, Nielsen, Cole, Cash, Jennifer N., Hall, James, Zhang, Ping, Simon, Sanford M., Cianfrocco, Michael A., and Taylor, Susan S. Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma. United States: N. p., 2020.
Web. doi:10.1371/journal.pbio.3001018.
Lu, Tsan-Wen, Aoto, Phillip C., Weng, Jui-Hung, Nielsen, Cole, Cash, Jennifer N., Hall, James, Zhang, Ping, Simon, Sanford M., Cianfrocco, Michael A., & Taylor, Susan S. Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma. United States. https://doi.org/10.1371/journal.pbio.3001018
Lu, Tsan-Wen, Aoto, Phillip C., Weng, Jui-Hung, Nielsen, Cole, Cash, Jennifer N., Hall, James, Zhang, Ping, Simon, Sanford M., Cianfrocco, Michael A., and Taylor, Susan S. Mon .
"Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma". United States. https://doi.org/10.1371/journal.pbio.3001018. https://www.osti.gov/servlets/purl/1903844.
@article{osti_1903844,
title = {Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma},
author = {Lu, Tsan-Wen and Aoto, Phillip C. and Weng, Jui-Hung and Nielsen, Cole and Cash, Jennifer N. and Hall, James and Zhang, Ping and Simon, Sanford M. and Cianfrocco, Michael A. and Taylor, Susan S.},
abstractNote = {When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.},
doi = {10.1371/journal.pbio.3001018},
journal = {PLoS Biology (Online)},
number = 12,
volume = 18,
place = {United States},
year = {Mon Dec 28 00:00:00 EST 2020},
month = {Mon Dec 28 00:00:00 EST 2020}
}
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