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Contributed by G. Marius Clore, October 15, 2020 (sent for review September 28, 2020; reviewed by Hashim M. Al-Hashimi and Lewis E. Kay)
Significance
Some of the most potent chaperones that protect against protein aggregation form heterogeneous oligomers that are crucial for function. Due to the polydisperse nature of these oligomers, the molecular origins that drive self-assembly remain poorly understood. Here, using a predominantly monomeric construct of the C-terminal domain of DNAJB6b we reveal an interplay between two N-terminal β strand configurations which we analyze quantitatively in structural and kinetic terms. We show that the two backbone conformations impact the assembly state of the C-terminal domain, with one being essentially monomeric and the other prone to oligomerization. The same residues in full-length DNAJB6b are vital for antiaggregation activity and thus we hypothesize that the equilibria characterized here lie at the core of DNAJB6b function.
Abstract
Chaperone oligomerization is often a key aspect of their function. Irrespective of whether chaperone oligomers act as reservoirs for active monomers or exhibit a chaperoning function themselves, understanding the mechanism of oligomerization will further our understanding of how chaperones maintain the proteome. Here, we focus on the class-II Hsp40, human DNAJB6b, a highly efficient inhibitor of protein self-assembly in vivo and in vitro that forms functional oligomers. Using single-quantum methyl-based relaxation dispersion NMR methods we identify critical residues for DNAJB6b oligomerization in its C-terminal domain (CTD). Detailed solution NMR studies on the structure of the CTD showed that a serine/threonine-rich stretch causes a backbone twist in the N-terminal β strand, stabilizing the monomeric form. Quantitative analysis of an array of NMR relaxation-based experiments (including Carr–Purcell–Meiboom–Gill relaxation dispersion, off-resonance R1ρ profiles, lifetime line broadening, and exchange-induced shifts) on the CTD of both wild type and a point mutant (T142A) within the S/T region of the first β strand delineates the kinetics of the interconversion between the major twisted-monomeric conformation and a more regular β strand configuration in an excited-state dimer, as well as exchange of both monomer and dimer species with high-molecular-weight oligomers. These data provide insights into the molecular origins of DNAJB6b oligomerization. Further, the results reported here have implications for the design of β sheet proteins with tunable self-assembling properties and pave the way to an atomic-level understanding of amyloid inhibition.
- Hsp40 chaperones
- relaxation-based NMR
- short-lived excited states
- conformational transitions
- oligomerization
Footnotes
↵1Present address: Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, LS2 9JT Leeds, United Kingdom.
- ↵2To whom correspondence may be addressed. Email: mariusc{at}mail.nih.gov.
Author contributions: T.K.K., V.T., and G.M.C. designed research, performed research, analyzed data, and wrote the paper.
Reviewers: H.M.A., Duke University Medical Center; and L.E.K., University of Toronto.
The authors declare no competing interest.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2020306117/-/DCSupplemental.
Data Availability.
Atomic coordinates, experimental restraints, and chemical shift assignments reported in this paper have been deposited in the Protein Data Bank, http://www.wwpdb.org/ (PDB ID code 7JSQ) (47). All the experimental relaxation-based NMR data shown in the SI Appendix and used to fit the kinetic models have also been deposited in digital format in Figshare (DOI: 10.6084/m9.figshare.13012682).
Published under the PNAS license.
References
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An S/T motif controls reversible oligomerization of the Hsp40 chaperone DNAJB6b through subtle reorganization of a β sheet backbone
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- Biological Sciences
- Biophysics and Computational Biology
- Physical Sciences
- Biophysics and Computational Biology
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