Regulatory inter-domain interactions influence Hsp70 recruitment to the DnaJB8 chaperone

Abstract

The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

“…4A). We observed disappearance of signals present in free hISG15 and emergence of new ones; this indicates slow-exchange binding regime 20,21 consistent with the sub-μM K d values measured by MST.…”

“…To gain insights into the PLpro CoV-2 :substrate contacts, we employed a combination of three cross-linkers that can link different moieties on protein surface. Disuccinimidyl suberate (DSS) and 4-(4,6 dimethoxy-1,3,5-triazin-2-yl)-4methyl-morpholinium chloride (DMTMM) allow detection of lysine-lysine and acid-lysine contacts, respectively 21,22 . We additionally used a newly developed hetero-bifunctional cross-linker that combines succinimide reactive group and a sulfonyl fluoride (SuFEx) which link lysines with amino acids containing hydroxyls but also lysines and histidines 23 .…”

“…The copyright holder for this preprint this version posted September 16, 2021. ; https://doi.org/10.1101/2021.09.15.460543 doi: bioRxiv preprint 21 37 °C while shaking at 600 rpm. 2% (v/v) formic acid was added to acidify the reaction and further purified by reversed-phase Sep-Pak tC18 cartridges (Waters), next flash frozen in liquid nitrogen and lyophilized.…”

Biophysical properties of a tau seed

“…The DLS experiments were performed similar to as described previously 31 . Reactions were prepared using the same experimental conditions as the ThT fluorescence aggregation experiments.…”

“…The SEC-MALS experiments were performed similar to as described previously 31 . 2 mg/ml of M i alone and in complex with 1:1 molar ratio of heparin (AMSbio, AMS.LMW Heparin) were filtered through a 0.22 μm PES filter before 100 μL each was applied to a Superdex 200 Increase 10/300 column (GE) equilibrated in 1xPBS (sigma 45ZP17) with 1 mM TCEP.…”