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The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and NMR spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a β-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events. Phosphorylation at a specific tyrosine by C-terminal Src kinase inactivates c-Src, whereas autophosphorylation is essential for the c-Src activation process. However, the structural consequences of the autophosphorylation process still remain elusive. Here we investigate how the structural landscape of c-Src is shaped by nucleotide binding and phosphorylation of Tyr 416 using biochemical experiments, hydrogen/deuterium exchange MS, and atomistic molecular simulations. We show that the initial steps of kinase activation involve large rearrangements in domain orientation. The kinase domain is highly dynamic and has strong cross-talk with the regulatory domains, which are displaced by autophosphorylation. Although the regulatory domains become more flexible and detach from the kinase domain because of autophosphorylation, the kinase domain gains rigidity, leading to stabilization of the ATP binding site and a 4-fold increase in enzymatic activity. Our combined results provide a molecular framework of the central steps in c-Src kinase regulation process with possible implications for understanding general kinase activation mechanisms., (© 2019 Boczek et al.)