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Open Access Publications from the University of California

Mechanistic insights into Hsp90 client engagement and remodeling through ATP hydrolysis

  • Author(s): Betegon Ramiro, Miguel
  • Advisor(s): Agard, David A
  • et al.

The molecular chaperone Hsp90 interacts with around 10% of the proteome, facilitating folding and regulating the biological function of its clients. Decades of work have led to a general understanding of the ATP-driven conformational cycle of Hsp90 and its role in client regulation, however the molecular details of this mechanism remain unknown. This is due in part to the intrinsic instability of Hsp90 clients, which has until recently hampered in-vitro and structural work, as well as to the difficulty in breaking down Hsp90s ATP-driven cycle into discrete steps that can be observed and measured together with changes in client structure and function. The work described in this thesis addresses two of the main questions that the Hsp90 field faces: 1) How is Hsp90’s conformational cycle and ATP hydrolysis used to remodel clients and 2) is there a conserved mechanism of client recognition and engagement that allows Hsp90 to functionally interact with dozens of clients of different size, sequence and structure.

The first chapter of this thesis addresses the first question and builds on work from Laura Lavery and James Partridge which revealed that the closed state of the mitochondrial Hsp90, TRAP1, is structurally asymmetric with one protomer in a ‘straight’ conformation while the other one adopts a novel ‘buckled’ conformation. Starting from this catalytic closed state, we investigate the effect of each ATP hydrolysis event on Hsp90’s conformation and how Hsp90 uses these hydrolysis-driven changes to remodel and regulate clients. We show that this asymmetry sets up sequential and deterministic hydrolysis, with the buckled protomer hydrolyzing first followed by a flip of the asymmetry that positions the unhydrolyzed protomer in the hydrolysis-favorable buckled conformation, promoting the second hydrolysis leading to reopening of the chaperone. While this asymmetry has not been observed in eukaryotic Hsp90 homologs, we propose that, despite being homodimers, all Hsp90s work as asymmetric machines, either through a structural asymmetry like in the case of TRAP1 or through formation of asymmetric complexes with clients and co-chaperones in the case of eukaryotic Hsp90s.

The third chapter of this thesis addresses the second question through the determination of the cryoEM structure of the bacterial Hsp90, HtpG, in complex with its client ribosomal protein L2. Though this work is still ongoing, the current cryoEM reconstruction of the apo-HtpG:L2 complex shows that, surprisingly, HtpG is in a GRP94-like conformation. Also striking is the fact that L2 interacts with HtpG in a Cdk4-like fashion, with density observed on both sides of HtpG and connected by a thread of density going through the lumen of HtpG along the same region previously identified for L2 and the model client Δ131Δ binding to HtpG. Additionally, L2 makes further interactions with HtpG different from those observed with Cdk4, notably with the N-terminal domain of HtpG around the lid area.

Overall, this work provides further evidence supporting that the different conformational states of Hsp90 are conserved across homologs but differentially regulated, and supports a conserved mechanism of interaction between Hsp90 and different classes of clients where Hsp90 clamps between domains stabilizing structural and likely short-lived transitions in the client.

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