Effects of Inhibitors on Hsp90′s Conformational Dynamics, Cochaperone and Client Interactions

Abstract

The molecular chaperone and heat-shock protein Hsp90 has become a central target in anti-cancer therapy. Nevertheless, the effect of Hsp90 inhibition is still not understood at the molecular level, preventing a truly rational drug design. Here we report on the effect of the most prominent drug candidates, namely, radicicol, geldanamycin, derivatives of purine, and novobiocin, on Hsp90's characteristic conformational dynamics and the binding of three interaction partners. Unexpectedly, the global opening and closing transitions are hardly affected by Hsp90 inhibitors. Moreover, we find no significant changes in the binding of the cochaperones Aha1 and p23 nor of the model substrate Δ131Δ. This holds true for competitive and allosteric inhibitors. Therefore, direct inhibition mechanisms affecting only one molecular interaction are unlikely. We suggest that the inhibitory action observed in vivo is caused by a combination of subtle effects, which can be used in the search for novel Hsp90 inhibition mechanisms.

Controlling protein function by fine-tuning conformational flexibility

“…We used the Single-Molecule Analysis of Complex Kinetic Sequences (SMACKS [ Schmid et al, 2016 ]) to quantify rate constants and uncertainties directly from the smFRET raw data. For Hsp90’s global conformational changes, we consistently infer 4-state models ( Schmid et al, 2016 ; Schmid et al, 2018 ; Schmid and Hugel, 2018 ): two low-FRET states (open conformations) and two high-FRET states (closed conformations). Although only two different FRET efficiencies can be resolved, at least four kinetic states are needed to describe the observed kinetic heterogeneity.…”

“…Surprisingly, the characteristic conformational changes of Hsp90 are only little affected by e.g. anti-cancer drug candidates ( Schmid et al, 2018 ) or natural nucleotides ( Schmid et al, 2016 ). In addition, to the stress-induced isoform discussed herein (Hsp82), there is also a cognate isoform (Hsc82) in yeast, which differs in unfolding stability, client range and more, despite 97% sequence identity ( Girstmair et al, 2019 ).…”

Molecular insights into the interaction of Hsp90 with allosteric inhibitors targeting the C-terminal domain

“…Therefore, similarly to novobiocin or the activating ligands, KU-32 and KU-596 binding to Hsp90 elicit global conformational changes to the Hsp90 dimer. Since apo-Hsp90α was utilised in the present study, this ligand-induced structural rearrangement may represent a shift to a novel N-Hsp90 dimerized state, which is distinct from the AMP-PNP stabilized state, 45 or a redistribution of conformations in the reorientation space sampled by the NM-Hsp90 domains of the two protomers, or may simply reflect an intra-protomer N-/C-Hsp90 communication relayed through M-Hsp90.…”

Hsp90 interaction networks in fungi—tools and techniques

“… 1997 ; Martin et al . 2008 ), they appear to exert multiple subtle effects that in combination reduce Hsp90 function and may cause small off-target effects (Schmid, Götz and Hugel 2018 ). Genetic depletion of Hsp90 is possible by producing mutants where Hsp90 is under a repressible promoter such as the tetracycline promoter (Gari et al .…”