Nucleotide-dependent domain interactions of Aha1-type co-chaperones with Hsp90 reveal evolutionarily conserved binding determinants.

Hsp90 is a dimeric molecular chaperone essential for the maturation, activation, stabilization, and folding of numerous clients required for cellular functions. Hsp90 progresses through a dynamic ATP-driven conformational cycle that is precisely regulated by accessory proteins known as co-chaperones. Here, we show that the isolated N-domain of Aha1 (Aha1N156) binds the apo state of Hsp90 but fails to associate with the closed, nucleotide-bound state. In contrast, the full-length Aha1 binds Hsp90 in both conformational states, suggesting a key role for the Aha1 C domain in binding to the nucleotide-bound, closed state of Hsp90. Surprisingly, the Aha1 paralogue Hch1, which corresponds to the Aha1 N domain, was capable of binding to Hsp90 in both the apo and nucleotide-bound states. Interestingly, the addition of a 14 amino acid residues section of the linker to the Aha1 N domain restores closed-state binding, indicating an unexpected role for the linker in stabilizing nucleotide-dependent interactions. Analysis of yeast-human Aha1 chimeras further demonstrates that the C-terminal domain of Aha-type co-chaperones serves as an evolutionarily conserved anchoring module, enabling stable engagement of the ATP-bound state despite significant sequence divergence. This work allows us to propose a model in which the Aha1 C domain allows for the repositioning of the Aha1 N domain that occurs during the transition from the apo to the ATP-bound state of Hsp90.