Distinct Phosphorylation Mechanisms as Dynamic Switches for Hsp90 Regulation

Phosphorylation, a critical post-translational modification, modulates protein function on demand without altering the genetic code. In Hsp90, an essential ATP-dependent molecular chaperone, phosphorylation plays a pivotal regulatory role, though the mechanisms underlying this regulation are not fully understood. Using solution NMR spectroscopy, we examined the conformational dynamics of Hsp90 at atomic resolution throughout its chaperone cycle. Our findings reveal distinct, phosphorylation-specific pathways that regulate Hsp90 activity. The phospho-mimetic mutation T115E induces widespread structural and dynamic perturbations across the N-terminal domain, enhancing flexibility while destabilizing key conformations. In contrast, T36E causes localized dynamic changes without significant structural perturbations, potentially disrupting the optimal conformational sampling required for efficient ATP hydrolysis. These distinct yet convergent pathways reshape the chaperone's dynamic landscape, enabling fine-tuned activity regulation. Our study underscores Hsp90's intrinsic plasticity in ATPase activity and establishes phosphorylation as a critical modulator within its functional network.