An S/T motif controls reversible oligomerization of the Hsp40 chaperone DNAJB6b through subtle reorganization of a β sheet backbone

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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. 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.
Funding Information
  • National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK-029023-20)

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