Coordinated ATP hydrolysis by the Hsp90 dimer

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Heat shock protein 90 (Hsp90) is a molecular chaperone.
• Hsp90 possesses a unique N-terminal ATP binding site, classifying it within the GHKL family of ATP-hydrolyzing enzymes.
• Structural data on ATP-induced conformational changes are available for some GHKL family members but lacking for Hsp90.

Purpose of the Study:

• To investigate the correlation between dimerization and ATP hydrolysis in Hsp90.
• To elucidate the role of N-terminal domains in Hsp90's ATPase activity and conformational changes.

Main Methods:

• Determination of the dimerization constant for wild-type (WT) Hsp90.
• Formation and analysis of heterodimers between WT Hsp90 and Hsp90 fragments or mutants lacking ATP binding domains.
• Measurement of ATPase activity in various Hsp90 constructs and heterodimers.

Main Results:

• WT Hsp90 has a dimerization constant of 60 nm.
• Heterodimers formed with ATP-binding domain-lacking fragments showed similar dimerization constants but reduced ATPase activity compared to WT Hsp90.
• A point mutant unable to bind ATP, when forming heterodimers with WT Hsp90, retained WT ATPase activity, suggesting ATP binding is not required for inter-domain stimulation.

Conclusions:

• Cooperative interactions within the N-terminal domains of Hsp90 are crucial for activating its ATPase activity.
• Dimerization and N-terminal domain contact formation may not strictly depend on ATP binding to both Hsp90 protomers.
• A mechanism is proposed where ATP hydrolysis is coupled to the opening-closing conformational movements of the Hsp90 chaperone.