The switch from client holding to folding in the Hsp70/Hsp90 chaperone machineries is regulated by a direct interplay between co-chaperones.

Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here, we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfold a stringent client, the glucocorticoid receptor ligand-binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. Current concepts assume that to proceed to client folding, Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 functionally interacts with Hop, relieves the stalling Hsp90-Hop interaction, and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle.

Structural elements in the flexible tail of the co-chaperone p23 coordinate client binding and progression of the Hsp90 chaperone cycle.

The co-chaperone p23 is a central part of the Hsp90 machinery. It stabilizes the closed conformation of Hsp90, inhibits its ATPase and is important for client maturation. Yet, how this is achieved has remained enigmatic. Here, we show that a tryptophan residue in the proximal region of the tail decelerates the ATPase by allosterically switching the conformation of the catalytic loop in Hsp90. We further show by NMR spectroscopy that the tail interacts with the Hsp90 client binding site via a conserved helix. This helical motif in the p23 tail also binds to the client protein glucocorticoid receptor (GR) in the free and Hsp90-bound form. In vivo experiments confirm the physiological importance of ATPase modulation and the role of the evolutionary conserved helical motif for GR activation in the cellular context.

The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma.

The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.

Author Correction: A methylated lysine is a switch point for conformational communication in the chaperone Hsp90.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90.

The heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.

A methylated lysine is a switch point for conformational communication in the chaperone Hsp90.

Methylation of a conserved lysine in C-terminal domain of the molecular chaperone Hsp90 was shown previously to affect its in vivo function. However, the underlying mechanism remained elusive. Through a combined experimental and computational approach, this study shows that this site is very sensitive to sidechain modifications and crucial for Hsp90 activity in vitro and in vivo. Our results demonstrate that this particular lysine serves as a switch point for the regulation of Hsp90 functions by influencing its conformational cycle, ATPase activity, co-chaperone regulation, and client activation of yeast and human Hsp90. Incorporation of the methylated lysine via genetic code expansion specifically shows that upon modification, the conformational cycle of Hsp90 is altered. Molecular dynamics simulations including the methylated lysine suggest specific conformational changes that are propagated through Hsp90. Thus, methylation of the C-terminal lysine allows a precise allosteric tuning of Hsp90 activity via long distances.

Coordinated Conformational Processing of the Tumor Suppressor Protein p53 by the Hsp70 and Hsp90 Chaperone Machineries.

p53, the guardian of the genome, requires chaperoning by Hsp70 and Hsp90. However, how the two chaperone machineries affect p53 conformation and regulate its function remains elusive. We found that Hsp70, together with Hsp40, unfolds p53 in an ATP-dependent reaction. This unfolded state of p53 is susceptible to aggregation after release induced by the nucleotide exchange factor Bag-1. However, when Hsp90 and the adaptor protein Hop are present, p53 is transferred from Hsp70 to Hsp90, allowing restoration of the native state upon ATP hydrolysis. Our results suggest that the p53 conformation is constantly remodeled by the two major chaperone machineries. This connects p53 activity to stress, and the levels of free molecular chaperones are important factors regulating p53 activity. Together, our findings reveal an intricate interplay and cooperation of Hsp70 and Hsp90 in regulating the conformation of a client.

Cytosolic Hsp70 and Hsp40 chaperones enable the biogenesis of mitochondrial ß-barrel proteins.

Mitochondrial ß-barrel proteins are encoded in the nucleus, translated by cytosolic ribosomes, and then imported into the organelle. Recently, a detailed understanding of the intramitochondrial import pathway of ß-barrel proteins was obtained. In contrast, it is still completely unclear how newly synthesized ß-barrel proteins reach the mitochondrial surface in an import-competent conformation. In this study, we show that cytosolic Hsp70 chaperones and their Hsp40 cochaperones Ydj1 and Sis1 interact with newly synthesized ß-barrel proteins. These interactions are highly relevant for proper biogenesis, as inhibiting the activity of the cytosolic Hsp70, preventing its docking to the mitochondrial receptor Tom70, or depleting both Ydj1 and Sis1 resulted in a significant reduction in the import of such substrates into mitochondria. Further experiments demonstrate that the interactions between ß-barrel proteins and Hsp70 chaperones and their importance are conserved also in mammalian cells. Collectively, this study outlines a novel mechanism in the early events of the biogenesis of mitochondrial outer membrane ß-barrel proteins.