Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones

The in vivo function of the heat shock protein 90 (Hsp90) molecular chapero ne is dependent on the binding and hydrolysis of ATP, and on interactions w ith a variety of co-chaperones containing tetratricopeptide repeat (TPR) do mains. We have now analysed the interaction of the yeast TPR-domain co-chap erones Sti1 and Cpr6 crith yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and de termined the effect of their binding on the inherent ATPase activity of Hsp 90, Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules b ind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solutio n. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATP ase activity by displacing Sti1 from Hsp90, Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain o f Hsp90, These results reveal an important role for TPR-domain co-chaperone s as regulators of the ATPase activity of Hsp90, showing that the ATP-depen dent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client -protein release.