Page, A.; Hendricks, W.; Dunckley, A.; Walti, M. A.

The human chaperone complex Hsp60/Hsp10 is essential for maintaining cellular proteostasis by preventing protein misfolding and aggregation. Disruption of these processes contributes to neurodegenerative diseases, while overexpression of Hsp60 and Hsp10 is associated with various cancers. Understanding their molecular mechanisms is therefore of fundamental importance. Unlike its bacterial homolog GroEL, human Hsp60 adopts multiple oligomeric states, with both heptameric and tetradecameric forms binding Hsp10 to form a folding cavity for substrate refolding. Here, we determine the cryo-EM structure of apo Hsp10 and find that, in addition to its single-ring form, it also assembles into a compact double-ring state. This reveals that Hsp10, like Hsp60, exhibits structural behavior that differs markedly from its bacterial counterpart. Traditionally viewed as a passive cofactor assisting Hsp60, we show that Hsp10 alone possesses intrinsic chaperone activity: it supports folding of natural substrates such as malate dehydrogenase 1 and manganese superoxide dismutase. NMR analysis further shows that substrate binding occurs primarily at the core of Hsp10 rather than at the loops. Our findings suggest that Hsp10 exists in equilibrium between single- and double-ring complexes in the unbound state, and upon binding as a single-ring complex, it actively guides substrates into the folding chamber.