ABSTRACT
In living organisms, most proteins work in complexes to form multicomponent protein machines. The function of such multicomponent machines is usually addressed by dividing them into a collection of two state systems at equilibrium. Many molecular machines, like Hsp90, work far from equilibrium by utilizing the energy of ATP hydrolysis. In these cases, important information is gained from the observation of the succession of more than two states in a row. We developed a four-colour single-molecule FRET system to observe the succession of states in the heat shock protein 90 (Hsp90) system, consisting of an Hsp90 dimer, the cochaperone p23 and nucleotides. We show that this multicomponent system is a directional ATP-dependent machinery. This reveals a previously undescribed mechanism on how cochaperones can modify Hsp90, namely by strengthening of the coupling between ATP hydrolysis and a kinetic step involved in the Hsp90 system resulting in a stronger directionality.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Multiprotein Complexes/metabolism , Phosphoproteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Adenosine Triphosphate/metabolism , Cloning, Molecular , Dimerization , Escherichia coli , Fluorescence Polarization , Fluorescent Dyes , Molecular Chaperones/genetics , Phosphoproteins/genetics , Schizosaccharomyces pombe Proteins/genetics , YeastsABSTRACT
The molecular chaperone heat shock protein 90 (Hsp90) is an important and abundant protein in eukaryotic cells, essential for the activation of a large set of signal transduction and regulatory proteins. During the functional cycle, the Hsp90 dimer performs large conformational rearrangements. The transient N-terminal dimerization of Hsp90 has been extensively investigated, under the assumption that the C-terminal interface is stably dimerized. Using a fluorescence-based single molecule assay and Hsp90 dimers caged in lipid vesicles, we were able to separately observe and kinetically analyze N- and C-terminal dimerizations. Surprisingly, the C-terminal dimer opens and closes with fast kinetics. The occupancy of the unexpected C-terminal open conformation can be modulated by nucleotides bound to the N-terminal domain and by N-terminal deletion mutations, clearly showing a communication between the two terminal domains. Moreover our findings suggest that the C- and N-terminal dimerizations are anticorrelated. This changes our view on the conformational cycle of Hsp90 and shows the interaction of two dimerization domains.
