N-terminal domain of human Hsp90 triggers binding to the cochaperone p23

Abstract

The molecular chaperone Hsp90 is a protein folding machine that is conserved from bacteria to man. Human, cytosolic Hsp90 is dedicated to folding of chiefly signal transduction components. The chaperoning mechanism of Hsp90 is controlled by ATP and various cochaperones, but is poorly understood and controversial. Here, we characterized the Apo and ATP states of the 170-kDa human Hsp90 full-length protein by NMR spectroscopy in solution, and we elucidated the mechanism of the inhibition of its ATPase by its cochaperone p23. We assigned isoleucine side chains of Hsp90 via specific isotope labeling of their δ-methyl groups, which allowed the NMR analysis of the full-length protein. We found that ATP caused exclusively local changes in Hsp90's N-terminal nucleotide-binding domain. Native mass spectrometry showed that Hsp90 and p23 form a 2∶2 complex via a positively cooperative mechanism. Despite this stoichiometry, NMR data indicated that the complex was not fully symmetric. The p23-dependent NMR shifts mapped to both the lid and the adenine end of Hsp90's ATP binding pocket, but also to large parts of the middle domain. Shifts distant from the p23 binding site reflect p23-induced conformational changes in Hsp90. Together, we conclude that it is Hsp90's nucleotide-binding domain that triggers the formation of the Hsp90 2 p23 2 complex. We anticipate that our NMR approach has significant impact on future studies of full-length Hsp90 with cofactors and substrates, but also for the development of Hsp90 inhibiting anticancer drugs.methyl-transverse-relaxation optimized spectroscopy | protein-protein interactions | heat shock proteins | asymmetry | allostery

Heterogeneity and dynamics in the assembly of the Heat Shock Protein 90 chaperone complexes

“…Although exemplified with part of the Hsp90 reaction cycle, the approach detailed here is applicable to other protein systems and is particularly suited (27). Hsp90 revealed well resolved charge state series corresponding to monomeric and predominantly dimeric Hsp90, in line with previous MS studies (28,29) (SI Appendix, Fig. S1A).…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…Chemical shift mapping revealed that the co-chaperone p23, which stabilizes Hsp90-substrate complexes [182], binds to both the N-and the M-domain of Hsp90 in a 2:2 ratio [193,194]. This binding interface is also used by another co-chaperone, Aha1, which binds preferentially in an asymmetric way to the closed ATP-dimer, as revealed by chemical shift perturbations and biophysical analysis, leading to the closed form of Hsp90 [195,196].…”

Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery

“…2b). According to X-ray crystallography 14 , isothermal titration calorimetry 34 , NMR and native mass spectrometry 35 we should be able to obtain a high acceptor (orange) signal for binding of p23 to Hsp90 and a low signal for unbinding. Indeed, Fig.…”