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Kirschke, E.

Publications and source records attributed to Kirschke, E..

2 recordsLinked to original sources
bioRxiv2020

GR chaperone cycle mechanism revealed by cryo-EM: inactivation of GR by GR:Hsp90:Hsp70:Hop client-loading complex

Maintaining a healthy proteome is fundamental for organism survival1,2. Integral to this are Hsp90 and Hsp70 molecular chaperones that together facilitate the folding, remodeling and maturation of Hsp90s many "client" proteins3-7. The glucocorticoid receptor (GR) is a model client strictly dependent upon Hsp90/Hsp70 for activity8-13. Chaperoning GR involves a cycle of inactivation by Hsp70, formation of an inactive GR:Hsp90:Hsp70:Hop "loading" complex, conversion to an active GR:Hsp90:p23 "maturation" complex, and subsequent GR release14. Unfortunately, a molecular understanding of this intricate chaperone cycle is lacking for any client. Here, we report the cryo-EM structure of the GR loading complex, in which Hsp70 loads GR onto Hsp90, revealing the molecular basis of direct Hsp90/Hsp70 coordination. The structure reveals two Hsp70s--one delivering GR and the other scaffolding Hop. Unexpectedly, the Hop cochaperone interacts with all components of the complex including GR, poising Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized via an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR maturation complex (Noddings et al., 2020), we present the first complete molecular mechanism of chaperone-dependent client remodeling, establishing general principles of client recognition, inhibition, transfer and activation.

biochemistry↗
bioRxiv2020

The interplay between Bag-1, Hsp70, and Hsp90 reveals that inhibiting Hsp70 rebinding is essential for Glucocorticoid Receptor activity

The glucocorticoid receptor (GR), like many signaling proteins requires Hsp90 for sustained activity. Previous biochemical studies revealed that the requirement for Hsp90 is explained by its ability to reverse Hsp70-mediated inactivation of GR through a complex process requiring both cochaperones and Hsp90 ATP hydrolysis. How ATP hydrolysis on Hsp90 enables GR reactivation is unknown. The canonical mechanism of client release from Hsp70 requires ADP:ATP exchange, which is normally rate limiting. Here we show that independent of ATP hydrolysis, Hsp90 acts as an Hsp70 nucleotide exchange factor (NEF) to accelerate ADP dissociation, likely coordinating GR transfer from Hsp70 to Hsp90. As Bag-1 is a canonical Hsp70 NEF that can also reactivate Hsp70:GR, the impact of these two NEFs was compared. Simple acceleration of Hsp70:GR release was insufficient for GR reactivation as Hsp70 rapidly re-binds and re-inactivates GR. Instead, inhibition of GR re-inactivation by Hsp70 is critical. This can be accomplished by high non-physiological Bag-1 concentrations, which also inhibit Hsp70:ATP binding. In contrast, in an ATP-hydrolysis dependent process, Hsp90 plays a unique role by kinetically partitioning GR into a state that can bind ligand, but is protected from Hsp70 inactivation, thus allowing GR to be activated by its ligand but still able to re-enter the chaperone cycle. At physiologic concentrations, Bag-1 works synergistically with Hsp90 to accelerate the first rate-limiting step in GR reactivation. The net effect is that the chaperone machinery cyclically dictates the on and off rates for GR ligand, providing a timer controlling the persistence of activated GR. Significance StatementThe glucocorticoid receptor (GR) is an essential transcription regulatory factor. Like many signaling proteins, GR activity is regulated by two essential molecular chaperones: Hsp90 and Hsp70. Functioning like a toggle switch, Hsp70 first inactivates GR, and then Hsp90 reactivates GR in an Hsp90 ATP hydrolysis dependent manner. Here, an intricate set of biochemistry experiments uncover fundamental principles governing how these chaperone systems collaboratively regulate GR activity. While Hsp90 promotes GR release from Hsp70 by modulating Hsp70s nucleotide state, this occurs independently of Hsp90 ATP hydrolysis. Instead, ATP hydrolysis on Hsp90 facilitates a second essential reactivation step resulting in an Hsp90-bound GR state that protects GR from Hsp70 re-inactivation. A kinetic partitioning model best describes chaperone modulation of GRs activity.

biochemistry↗