C-terminal regions of Hsp90 are important for trapping the nucleotide during the ATPase cycle

Hsp90 is an abundant molecular chaperone that functions in an ATP-dependent manner in vivo. The ATP-binding site is located in the N-terminal domain o f Hsp90. Here, we dissect the ATPase cycle of Hsp90 kinetically. We find th at Hsp90 binds ATP with a two-step mechanism. The rate-limiting step of the ATPase cycle is the hydrolysis of ATP. Importantly, ATP becomes trapped an d committed to hydrolyze during the cycle. In the isolated ATP-binding doma in of Hsp90, however, the bound ATP was not committed and the turnover numb ers were markedly reduced. Analysis of a series of truncation mutants of Hs p90 showed that C-terminal regions far apart in sequence from the ATP-bindi ng domain are essential for trapping the bound ATP and for maximum hydrolys is rates. Our results suggest that ATP binding and hydrolysis drive conform ational changes that involve the entire molecule and lead to repositioning of the N and C-terminal domains of Hsp90. (C) 2000 Academic Press.