Insights into (un)structure-function relationships of intrinsically disordered chaperone Hsp33

Cells cope constantly with environmental challenges which have potentially fatal consequences. Redox and heat fluctuations cause stress that leads to protein unfolding, aggregation, and cell death. Different post-translational modifications affect the protein’s structure, function, and stability. Therefore, it is not surprising that evolution armed cells with a system of stress-regulated chaperones which maintains proteome health during stress conditions.

One such protein is the ATP-independent, redox-regulated, intrinsically disordered and highly conserved bacterial chaperone Hsp33. When oxidized, Hsp33 undergoes redox-dependent unfolding essential for anti-aggregation activity. Upon reduction, Hsp33 refolds and releases its client protein to the DnaK/J system for further refolding. Unlike other proteins, Hsp33 must lose its structure in order to gain function. The activation mechanism of Hsp33 is triggered by oxidation of the redox-sensitive cysteines located in the C-terminal domain, followed by destabilization of the adjacent metastable linker region harboring hydrophobic regions, which were proposed to serve as binding sites.

Replacement of the entire 50aa linker region by diverse non-native sequences created a set of constitutively active, super-holdase chaperones. Here we employ a toolbox of structural mass spectrometry techniques, including hydrogen-deuterium exchange (HDX) and crosslinking (XL) coupled with mass spectrometry to define substrate promiscuity of the conditionally disordered chaperone, Hsp33. We show that extensive sequence modification of the metastable region leads to displacement of the binding site, preserving tight binding with client proteins. This study demonstrates how protein plasticity can play a major role in protein-protein interaction and substrate promiscuity underlying chaperone and other multi-substrate proteins.