Heterogeneity and dynamics in the assembly of the heat shock protein 90 chaperone complexes.
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.
Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3TA, United Kingdom.
Ima-obong
IO
Ebong
Nina
N
Morgner
Min
M
Zhou
Marco A
MA
Saraiva
Soumya
S
Daturpalli
Sophie E
SE
Jackson
Carol V
CV
Robinson
D019281:Dimerization; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D011485:Protein Binding; D022021:Tacrolimus Binding Proteins / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; D022021:Tacrolimus Binding Proteins; C403485:tacrolimus binding protein 4