Defining the binding pattern of the disordered Hsp33 chaperone using an integrative structural approach

Cells constantly cope with environmental challenges, including oxidative stress, which has potentially fatal consequences on protein structure, function, and stability. Therefore, it is not surprising that evolution armed cells with a system of stress-regulated chaperones which maintain proteome health during stress conditions.

ATP-independent chaperones serve as the first line of defense against protein denaturation and aggregation by utilizing their structural plasticity induced by specific stress conditions. One such protein is the redox-regulated intrinsically disordered chaperone, Hsp33. When oxidized, Hsp33 undergoes redox-dependent unfolding essential for anti-aggregation activity. Unlike other proteins, Hsp33 must lose its structure to gain function.

Unfortunately, the exact role of the structural unfolding in the substrate recognition of Hsp33 is yet uncovered. Here we developed an integrative platform combining structural mass spectrometry techniques (HDX-MS and XL-MS) with computational modeling to define substrate promiscuity of Hsp33. We have identified binding hotspots of Hsp33, crucial for the recognition of its substrates. Moreover, we show that an extensive sequence alteration of the metastable region of Hsp33 results in a displacement of the binding sites of Hsp33. This study maps the redox-regulated cascade of structural rearrangements and multiple states of Hsp33, which define the role of protein plasticity in substrate promiscuity required for chaperone activity and recognition of numerous targets during oxidative stress. This study shows how our integrative methodology platform allows high-resolution mapping of protein dynamics and binding sites in structurally challenging proteins.