Abstract
The cochaperone Sti1/Hop physically links Hsp70 and Hsp90. The protein exhibits one binding site for Hsp90 (TPR2A) and two binding sites for Hsp70 (TPR1 and TPR2B). How these sites are used remained enigmatic. Here we show that Sti1 is a dynamic, elongated protein that consists of a flexible N-terminal module, a long linker and a rigid C-terminal module. Binding of Hsp90 and Hsp70 regulates the Sti1 conformation with Hsp90 binding determining with which site Hsp70 interacts. Without Hsp90, Sti1 is more compact and TPR2B is the high-affinity interaction site for Hsp70. In the presence of Hsp90, Hsp70 shifts its preference. The linker connecting the two modules is crucial for the interaction with Hsp70 and for client activation in vivo. Our results suggest that the interaction of Hsp70 with Sti1 is tightly regulated by Hsp90 to assure transfer of Hsp70 between the modules, as a prerequisite for the efficient client handover.
Highlights
The cochaperone Sti1/Hop physically links Hsp[70] and Hsp[90]
As previous studies showed that Sti1/Hop is a monomer[35], this indicates an elongated shape of Sti[1]
Solution-based single-pair Forster Resonance Energy Transfer (spFRET) experiments on constructs where the donor and acceptor molecules were fluorescently labelled near the C- and N-terminus of Sti[1] (S2C-G588C, S2CS523C or G131C-S523C) exhibited no appreciable FRET signal (Supplementary Fig. 1)
Summary
The cochaperone Sti1/Hop physically links Hsp[70] and Hsp[90]. The protein exhibits one binding site for Hsp[90] (TPR2A) and two binding sites for Hsp[70] (TPR1 and TPR2B). The linker connecting the two modules is crucial for the interaction with Hsp[70] and for client activation in vivo. Another intriguing feature of Sti[1] is that each of the two Hsp70-binding TPR domains is followed by a DP domain These DP domains, especially DP2 located C-terminally of the crucial TPR2B domain, are important for client activation in vivo[22,30,31,32]. Such a TPR–DP module is present in Hip (Hsp70-interacting protein), an Hsp[70] cochaperone[33,34]. Binding of Hsp[90] affects the interaction of Hsp[70] with the two potential binding sites in Sti[1]
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