Electron Paramagnetic Resonance (EPR) Spectroscopy Reveals Nucleotide- and Partner-Dependent Conformational Ensembles in Hsp90

Abstract

Heat shock protein 90 kDa (Hsp90) is an ATP-dependent molecular chaperone whose function relies on conformational rearrangements and interactions with cochaperones and clients. Binding of nucleotides, cochaperones, and clients leads to functional structural rearrangements, often associated with heterogeneous structural ensembles that are challenging to characterize using high-resolution structure determination methods alone. Electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach for tracking the conformational landscape of proteins. However, despite its great potential, its application to Hsp90 homologs has so far remained limited, and the insights gained have not yet been comprehensively reviewed. In this mini-review, we summarize EPR studies on cytosolic, mitochondrial, and endoplasmic reticulum Hsp90 with the aim of highlighting how EPR has advanced our understanding of nucleotide- and partner-dependent conformations. Specifically, we summarize how continuous wave EPR (CW-EPR), double electron–electron resonance (DEER), and electron nuclear double resonance (ENDOR) spectroscopies revealed conformational equilibria, symmetry breaking, dissociation constants, and the structural role of intrinsically disordered regions within Hsp90, while also discussing methodological advances and recent extensions to cellular contexts. Overall, these studies establish EPR spectroscopy as a valuable biophysical tool for resolving conformational ensembles and structural heterogeneity underlying Hsp90’s function, with general relevance to the structural biology, spectroscopy, and chaperone fields, and pave the way for future in vitro and in-cell investigations of this exciting molecular chaperone.