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

Abstract

The Hsp90 cycle depends on the coordinated activity of a range of cochaperones, including Hop, Hsp70 and peptidyl-prolyl isomerases such as FKBP52. Using mass spectrometry, we investigate the order of addition of these cochaperones and their effects on the stoichiometry and composition of the resulting Hsp90-containing complexes. Our results show that monomeric Hop binds specifically to the Hsp90 dimer whereas FKBP52 binds to both monomeric and dimeric forms of Hsp90. By preforming Hsp90 complexes with either Hop, followed by addition of FKBP52, or with FKBP52 and subsequent addition of Hop, we monitor the formation of a predominant asymmetric ternary complex containing both cochaperones. This asymmetric complex is subsequently able to interact with the chaperone Hsp70 to form quaternary complexes containing all four proteins. Monitoring the population of these complexes during their formation and at equilibrium allows us to model the complex formation and to extract 14 different K D values. This simultaneous calculation of the K D s from a complex system with the same method, from eight deferent datasets under the same buffer conditions delivers a self-consistent set of values. In this case, the K D values afford insights into the assembly of ten Hsp90-containing complexes and provide a rationale for the cellular heterogeneity and prevalence of intermediates in the Hsp90 chaperone cycle. dissociation constants | interaction network

Fungal Hsp90: a biological transistor that tunes cellular outputs to thermal inputs

“…Mass spectrometric analyses of complexes formed in vitro by recombinant human Hsp90, Hsp70, Hop, and FKBP52 allowed the authors to construct an interaction network for these factors and to predict dominant complexes that are formed during the Hsp90 cycle. The relative similarity of the binding constants for the species studied supported the view that the Hsp90 chaperone machinery is complex and dynamic 121 .…”

“…Ebong and colleagues analysed the kinetics of Hsp90 complex formation with its chaperones 121 . Mass spectrometric analyses of complexes formed in vitro by recombinant human Hsp90, Hsp70, Hop, and FKBP52 allowed the authors to construct an interaction network for these factors and to predict dominant complexes that are formed during the Hsp90 cycle.…”

Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop

“…NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6484 ARTICLE stabilizes the compact conformation of the ternary complex 21,32 . This interaction is not stable on its own since there is no binding between purified Hsp70 NBD and Hsp90 (results not shown), but it takes place in the compact conformation of the ternary complex and cannot be explained by the crosslinking, since we found particles in which Hsp70 did not contact Hsp90 (the 'extended' complexes) and conversely, we found compact ternary complex structures in the crosslinker-free AUC experiments (Supplementary Fig.…”

The Effect of Detergent, Temperature, and Lipid on the Oligomeric State of MscL Constructs: Insights from Mass Spectrometry

“…Because detergent removal is dictated by the physical properties of the detergent micelle (Reading et al, 2015), it is not possible to compare accelerating voltage conditions for constructs in different detergents. The relative abundances of oligomers were calculated using the UniDec deconvolution software program (Marty et al, 2015) with detector efficiency taken into account (Ebong et al, 2011;Fraser, 2002;Stengel et al, 2012).…”