Abstract
Hsp90 is an essential chaperone that requires large allosteric changes to determine its ATPase activity and client binding. The co‐chaperone Aha1, which is the major ATPase stimulator in eukaryotes, is important for regulation of Hsp90's allosteric timing. Little is known, however, about the structure of the Hsp90/Aha1 complex. Here, we characterize the solution structure of unmodified human Hsp90/Aha1 complex using NMR spectroscopy. We show that the 214‐kDa complex forms by a two‐step binding mechanism and adopts multiple conformations in the absence of nucleotide. Aha1 induces structural changes near Hsp90's nucleotide‐binding site, providing a basis for its ATPase‐enhancing activity. Our data reveal important aspects of this pivotal chaperone/co‐chaperone interaction and emphasize the relevance of characterizing dynamic chaperone structures in solution.
Highlights
Mapping of the affected isoleucines on the structure of Hsp9019 showed that the most strongly broadened residues are located at the interface between the middle (Hsp90M) and C-terminal domain (Hsp90C) of Hsp[90], as well as the N-terminal interface involved in dimer closure (Figure 1c and Figure S1b)
The regions affected in Hsp90M agree with the binding site of the N-terminal domain of Aha[1] (Aha1-N), as displayed in the crystal structure of the complex of yeast activator of Heat shock protein of 90 kDa (Hsp90) ATPase activity 1 (Aha1)-N with Hsp90M (Figure 1c,d).[21]
Hsp90N, which would interact with Aha1-C assuming a head-to-tail mode of interaction,[21] was more broadly affected (Figure 1c,d and Figure S2), in agreement with footprinting of the Hsp90/Aha1-interaction using crosslinking coupled to mass spectrometry,[12] but contrary to a previously proposed model built on the basis of individual Hsp[90] and Aha[1] domain constructions.[15]
Summary
51 kDa; Hsp[90], heat shock protein of 90 kDa; NMR, nuclear magnetic resonance; SAXS, small-angle X-ray scattering; TROSY, transverse relaxation-optimized spectroscopy.Heat shock protein of 90 kDa (Hsp90) is a highly conserved ATP-dependent molecular chaperone responsible for the stabilization, maturation, and activation of many client proteins.[1,2] Several co-chaperones regulate Hsp90's activation cycle, which is essential to maintain protein homeostasis.[3,4] During its activation cycle, the Hsp[90] dimer undergoes large conformationalProtein Science. 2019;28:1545–1551.wileyonlinelibrary.com/journal/pro 1545 OROZ ET AL.rearrangements from an extended to a closed, ATPase-active conformation, a process which involves intra- and interprotomer interactions.[5,6,7] Different Hsp[90] orthologs show distinct conformational equilibria[8] that are translated into different ATPase activities. Comparative analysis of perturbations observed in nuclear magnetic resonance (NMR) spectra indicate that Aha[1] promotes conformational rearrangements in Hsp[90] that favor ATP binding and hydrolysis.
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