Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis
Contributed by G. Marius Clore, September 4, 2019 (sent for review August 29, 2019; reviewed by Lewis E. Kay and James G. Omichinski)
Significance
Members of the Hsp40/DnaJ family are cochaperones of the Hsp70 cycle and, additionally, act independently to prevent protein aggregation. DNAJB6b inhibits amyloid formation but has eluded detailed structural analysis due to its polydisperse oligomeric nature. Here, using a predominantly monomeric deletion variant, we unravel the solution structure and dynamics of DNAJB6b by NMR. DNAJB6b consists of 2 domains, a Hsp70-binding J domain and a substrate-binding C-terminal domain, that tumble almost independently of each other. Transient (millisecond time scale) intramolecular interactions are observed between the 2 domains, as well as intermolecular interactions between the C-terminal domains resulting in the formation of large oligomers. The results provide insights into regulation of the Hsp70 cycle by Hsp40 and the mechanism of antiaggregation.
Abstract
J-domain chaperones are involved in the efficient handover of misfolded/partially folded proteins to Hsp70 but also function independently to protect against cell death. Due to their high flexibility, the mechanism by which they regulate the Hsp70 cycle and how specific substrate recognition is performed remains unknown. Here we focus on DNAJB6b, which has been implicated in various human diseases and represents a key player in protection against neurodegeneration and protein aggregation. Using a variant that exists mainly in a monomeric form, we report the solution structure of an Hsp40 containing not only the J and C-terminal substrate binding (CTD) domains but also the functionally important linkers. The structure reveals a highly dynamic protein in which part of the linker region masks the Hsp70 binding site. Transient interdomain interactions via regions crucial for Hsp70 binding create a closed, autoinhibited state and help retain the monomeric form of the protein. Detailed NMR analysis shows that the CTD (but not the J domain) self-associates to form an oligomer comprising ∼35 monomeric units, revealing an intricate balance between intramolecular and intermolecular interactions. The results shed light on the mechanism of autoregulation of the Hsp70 cycle via conserved parts of the linker region and reveal the mechanism of DNAJB6b oligomerization and potentially antiaggregation.
Data Availability
Data deposition: The data reported in this paper have been deposited in the Protein Data Bank www.wwpdb.org (PDB ID codes 6U3R and 6U3S [coordinates and restraints]) and Biological Magnetic Resonance Data Bank www.bmrb.wisc.edu (BMRB ID code 30656 [chemical shifts]).
Acknowledgments
We thank Drs. Charles Schwieters and Harm Kampinga for useful discussions; and Drs. James Baber, Dan Garrett, and Jinfa Ying for technical support. This work was funded by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (to G.M.C.).
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© 2019. Published under the PNAS license.
Data Availability
Data deposition: The data reported in this paper have been deposited in the Protein Data Bank www.wwpdb.org (PDB ID codes 6U3R and 6U3S [coordinates and restraints]) and Biological Magnetic Resonance Data Bank www.bmrb.wisc.edu (BMRB ID code 30656 [chemical shifts]).
Submission history
Published online: October 7, 2019
Published in issue: October 22, 2019
Keywords
Acknowledgments
We thank Drs. Charles Schwieters and Harm Kampinga for useful discussions; and Drs. James Baber, Dan Garrett, and Jinfa Ying for technical support. This work was funded by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (to G.M.C.).
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The authors declare no competing interest.
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Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis, Proc. Natl. Acad. Sci. U.S.A.
116 (43) 21529-21538,
https://doi.org/10.1073/pnas.1914999116
(2019).
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