Coordinated ATP hydrolysis by the Hsp90 dimer

The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP bin ding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrol ysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determ ined to be 60 nm. Heterodimers of WT Hsp90 with fragments lacking the ATP b inding domain form readily and exhibit dimerization constants similar to fu ll-length Hsp90. However, the ATPase activity of these heterodimers was sig nificantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activa tion of the ATPase activity. To further address the contribution of the N-t erminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this m utant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. T hus, contact formation between the N-terminal domains might not depend on A TP bound to both domains. Together, these results suggest a mechanism for c oupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone.