HSPB5 engages multiple states of a destabilized client to enhance chaperone activity in a stress-dependent manner.

Small heat shock proteins (sHSPs) delay protein aggregation in an ATP-independent manner by interacting with client proteins that are in states susceptible to aggregation, including destabilized states related to cellular stress. Up-regulation of sHSPs under stress conditions supports their critical role in cellular viability. Widespread distribution of sHSPs in most organisms implies conservation of function, but it remains unclear whether sHSPs implement common or distinct mechanisms to delay protein aggregation. Comparisons among various studies are confounded by the use of different model client proteins, different assays for both aggregation and sHSP/client interactions, and variable experimental conditions used to mimic cellular stress. To further define sHSP/client interactions and their relevance to sHSP chaperone function, we implemented multiple strategies to characterize sHSP interactions with α-lactalbumin, a model client whose aggregation pathway is well defined. We compared the chaperone activity of human αB-crystallin (HSPB5) with HSPB5 variants that mimic states that arise under conditions of cellular stress or disease. The results show that these closely related sHSPs vary not only in their activity under identical conditions but also in their interactions with clients. Importantly, under nonstress conditions, WT HSPB5 delays client aggregation solely through transient interactions early in the aggregation pathway, whereas HSPB5 mutants that mimic stress-activated conditions can also intervene at later stages of the aggregation pathway to further delay client protein aggregation.