Mechanism of small heat shock protein client sequestration and induced polydispersity.

Small heat shock proteins (sHSPs) act as first responders during cellular stress by recognizing and sequestering destabilized proteins (clients), preventing their aggregation and facilitating downstream refolding or degradation. This chaperone function is critically important to proteostasis, conserved across all kingdoms of life, and associated with various protein misfolding diseases in humans. Mechanistic insights into how sHSPs sequester destabilized clients have been limited due to the extreme molecular plasticity and client-induced polydispersity of sHSP/client complexes. Here, we present high-resolution cryo-EM structures of the sHSP from (HSP16.5) in both the apo-state and in an ensemble of client-bound states. The ensemble not only reveals key molecular mechanisms by which sHSPs respond to and sequester client proteins, but also provides insights into the cooperative nature of chaperone-client interactions. Engagement with destabilized client induces a polarization of stability across the HSP16.5 scaffold, proposed to facilitate higher-order assembly and enhance client sequestration capacity. Some higher-order sHSP oligomers appear to form through simple insertion of dimeric subunits into new geometrical features, while other higher-order states suggest multiple sHSP/client assembly pathways. Together, these results provide long-sought insights into the chaperone function of sHSPs and highlight the relationship between polydispersity and client sequestration under stress conditions.