Designed Hsp90 heterodimers reveal an asymmetric ATPase-driven mechanism in vivo.

Hsp90 is a homodimeric ATPase that is essential in eukaryotes for the maturation of client proteins frequently involved in signal transduction, including many kinases and nuclear steroid hormone receptors. Competitive inhibitors of ATP binding to Hsp90 prevent client maturation and show promise as anticancer agents in clinical trials. However, the role of ATP binding and hydrolysis in each subunit of the Hsp90 dimer has been difficult to investigate because of an inability to assemble and study dimers of defined composition. We used protein engineering to generate functional Hsp90 subunits that preferentially assemble as heterodimers. We analyzed dimers wherein one subunit harbors a disruptive mutation and observed that ATP binding by both subunits is essential for function in yeast, whereas ATP hydrolysis is only required in one subunit. These findings demonstrate important functional contributions from both symmetric and asymmetric Hsp90 dimers and provide valuable reagents for future investigations of Hsp90 mechanism.