Structural Insights into the Regulation Mechanism of HSP90 by Co-chaperone AHA1

Abstract

Heat Shock Protein 90 (Hsp90) is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of “client” proteins, especially those involved in signaling and regulatory processes. This Hsp90 chaperone machinery is also essential for maintaining cellular protein homeostasis and for dealing with proteotoxic stress. The client maturation cycle is regulated by a large number of co-chaperones that sequentially interact with Hsp90 throughout its ATPase cycle. One of the most important, but poorly understood co-chaperones in eukaryotes is Activator of Hsp90 ATPase homolog 1 (Aha1). As a potent stimulator of the Hsp90 ATPase activity, Aha1 is thought to accelerate client protein remodeling. A knockout of Aha1 in yeast severely compromises the maturation of different Hsp90 client proteins. However, Aha1 overexpression inhibits the rate of Hsp90-dependent refolding of denatured luciferase, as well as cystic fibrosis associated protein CFTR. How Aha1, as an Hsp90 activator, facilitates ATP hydrolysis while inhibiting client maturation remains enigmatic. To better understand this question, we have determined the structure of the full-length 200-kDa Hsp90/Aha1 complex at 3.8 Å resolution using cryo-electron microscopy. When bound to Aha1, the Hsp90 was captured in the closed conformation, similar to the X-ray crystal structure of Hsp90 bound with inhibitory co-chaperone p23. However, the binding interface is completely different from that used by p23 as well as that observed by crystallography of a fragment of Aha bound to a fragment of Hsp90. In our structure, the C-terminal domain of Aha1 stabilizes the Hsp90 closed dimer by bridging the C-terminal domain of one protomer and the middle domain/N-terminal domain of the other protomer. This structure begins to explain the Aha1 inhibitory effect on Hsp90 and also provides a basis for designing co-chaperone specific inhibitors to regulate Hsp90 related diseases.