Distinct phosphorylation mechanisms as dynamic switches for Hsp90 regulation

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein phosphorylation is a key post-translational modification regulating protein function.
• The molecular chaperone Heat Shock Protein 90 (Hsp90) is ATP-dependent and its activity is controlled by multiple phosphorylation sites.
• The precise mechanisms by which individual phosphorylation sites regulate Hsp90's function remain incompletely understood.

Purpose of the Study:

• To investigate how site-specific phosphorylation reshapes the conformational energy landscape of Hsp90.
• To elucidate the distinct regulatory roles of individual phosphorylation sites in Hsp90's ATPase cycle.

Main Methods:

• Solution Nuclear Magnetic Resonance (NMR) spectroscopy was employed to study Hsp90.
• Phospho-mimetic mutations (T115E and T36E) were introduced to mimic phosphorylation.
• Conformational dynamics and energy landscapes were analyzed across the ATPase cycle.

Main Results:

• The T115E mutation globally altered the N-terminal domain energy landscape, reducing barriers and affecting ATP/ADP binding states.
• The T36E mutation selectively impacted dynamics at the ATP-bound state, biasing the ensemble towards the ground state.
• Both mutations converged to suppress productive progression through the Hsp90 ATPase cycle.

Conclusions:

• Individual phosphorylation sites on Hsp90 utilize distinct allosteric pathways to regulate protein function.
• Phosphorylation acts as a crucial regulator, fine-tuning Hsp90's ATPase cycle through dynamic allosteric control.
• Understanding these site-specific regulatory mechanisms is vital for comprehending Hsp90's chaperone activity.