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.

NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.

The HSP90 chaperone machinery.

The heat shock protein 90 (HSP90) chaperone machinery is a key regulator of proteostasis under both physiological and stress conditions in eukaryotic cells. As HSP90 has several hundred protein substrates (or 'clients'), it is involved in many cellular processes beyond protein folding, which include DNA repair, development, the immune response and neurodegenerative disease. A large number of co-chaperones interact with HSP90 and regulate the ATPase-associated conformational changes of the HSP90 dimer that occur during the processing of clients. Recent progress has allowed the interactions of clients with HSP90 and its co-chaperones to be defined. Owing to the importance of HSP90 in the regulation of many cellular proteins, it has become a promising drug target for the treatment of several diseases, which include cancer and diseases associated with protein misfolding.

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.

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.

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2.

The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.

Biogenesis of secretory immunoglobulin M requires intermediate non-native disulfide bonds and engagement of the protein disulfide isomerase ERp44.

Antibodies of the immunoglobulin M (IgM) class represent the frontline of humoral immune responses. They are secreted as planar polymers in which flanking µ2 L2 "monomeric" subunits are linked by two disulfide bonds, one formed by the penultimate cysteine (C575) in the tailpiece of secretory µ chains (µs tp) and the second by C414 in the Cµ3. The latter bond is not present in membrane IgM. Here, we show that C575 forms a non-native, intra-subunit disulfide bond as a key step in the biogenesis of secretory IgM. The abundance of this unexpected intermediate correlates with the onset and extent of polymerization. The rearrangement of the C-terminal tails into a native quaternary structure is guaranteed by the engagement of protein disulfide isomerase ERp44, which attacks the non-native C575 bonds. The resulting conformational changes promote polymerization and formation of C414 disulfide linkages. This unusual assembly pathway allows secretory polymers to form without the risk of disturbing the role of membrane IgM as part of the B cell antigen receptor.

Identification of a chaperone-code responsible for Rad51-mediated genome repair.

Posttranslational modifications of Hsp90 are known to regulate its in vivo chaperone functions. Here, we demonstrate that the lysine acetylation-deacetylation dynamics of Hsp82 is a major determinant in DNA repair mediated by Rad51. We uncover that the deacetylated lysine 27 in Hsp82 dictates the formation of the Hsp82-Aha1-Rad51 complex, which is crucial for client maturation. Intriguingly, Aha1-Rad51 complex formation is not dependent on Hsp82 or its acetylation status; implying that Aha1-Rad51 association precedes the interaction with Hsp82. The DNA damage sensitivity of Hsp82 (K27Q/K27R) mutants are epistatic to the loss of the (de)acetylase hda1Δ; reinforcing the importance of the reversible acetylation of Hsp82 at the K27 position. These findings underscore the significance of the cross talk between a specific Hsp82 chaperone modification code and the cognate cochaperones in a client-specific manner. Given the pivotal role that Rad51 plays during DNA repair in eukaryotes and particularly in cancer cells, targeting the Hda1-Hsp90 axis could be explored as a new therapeutic approach against cancer.

The Plasticity of the Hsp90 Co-chaperone System.

The Hsp90 system in the eukaryotic cytosol is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function. Although progress has been made regarding the underlying biochemical mechanisms, how co-chaperones influence Hsp90 client proteins in vivo has remained elusive. By investigating the effect of 12 Hsp90 co-chaperones on the activity of different client proteins in yeast, we find that deletion of co-chaperones can have a neutral or negative effect on client activity but can also lead to more active clients. Only a few co-chaperones are active on all clients studied. Closely related clients and even point mutants can depend on different co-chaperones. These effects are direct because differences in client-co-chaperone interactions can be reconstituted in vitro. Interestingly, some co-chaperones affect client conformation in vivo. Thus, co-chaperones adapt the Hsp90 cycle to the requirements of the client proteins, ensuring optimal activation.

Active unfolding of the glucocorticoid receptor by the Hsp70/Hsp40 chaperone system in single-molecule mechanical experiments.

The glucocorticoid receptor (GR) is an important transcription factor and drug target linked to a variety of biological functions and diseases. It is one of the most stringent physiological clients of the Hsp90/Hsp70/Hsp40 chaperone system. In this study, we used single-molecule force spectroscopy by optical tweezers to observe the interaction of the GR's ligand-binding domain (GR-LBD) with the Hsp70/Hsp40 chaperone system (Hsp70/40). We show in real time that Hsp70/40 can unfold the complete GR-LBD in a stepwise manner. Each unfolding step involves binding of an Hsp70 to the GR-LBD and subsequent adenosine triphosphate (ATP) hydrolysis, stimulated by Hsp40. The kinetics of chaperone-mediated unfolding depend on chaperone concentrations as well as the presence of the nucleotide exchange factor BAG1. We find that Hsp70/40 can stabilize new unfolding intermediates, showing that Hsp70/40 can directly interact with the folded core of the protein when working as an unfoldase. Our results support an unfolding mechanism where Hsp70 can directly bind to folded protein structures and unfold them upon ATP hydrolysis. These results provide important insights into the regulation of GR by Hsp70/40.

The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions.

Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.

p23 and Aha1: Distinct Functions Promote Client Maturation.

Hsp90 is a conserved molecular chaperone regulating the folding and activation of a diverse array of several hundreds of client proteins. The function of Hsp90 in client processing is fine-tuned by a cohort of co-chaperones that modulate client activation in a client-specific manner. They affect the Hsp90 ATPase activity and the recruitment of client proteins and can in addition affect chaperoning in an Hsp90-independent way. p23 and Aha1 are central Hsp90 co-chaperones that regulate Hsp90 in opposing ways. While p23 inhibits the Hsp90 ATPase and stabilizes a client-bound Hsp90 state, Aha1 accelerates ATP hydrolysis and competes with client binding to Hsp90. Even though both proteins have been intensively studied for decades, research of the last few years has revealed intriguing new aspects of these co-chaperones that expanded our perception of how they regulate client activation. Here, we review the progress in understanding p23 and Aha1 as promoters of client processing. We highlight the structures of Aha1 and p23, their interaction with Hsp90, and how their association with Hsp90 affects the conformational cycle of Hsp90 in the context of client maturation.

Hsp90-mediated regulation of DYRK3 couples stress granule disassembly and growth via mTORC1 signaling.

Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual-specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.

The Chaperone Activity of the Developmental Small Heat Shock Protein Sip1 Is Regulated by pH-Dependent Conformational Changes.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains.

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody VL domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient VL domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient VL domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the VL domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.

Collagen's primary structure determines collagen:HSP47 complex stoichiometry.

Collagens play important roles in development and homeostasis in most higher organisms. In order to function, collagens require the specific chaperone HSP47 for proper folding and secretion. HSP47 is known to bind to the collagen triple helix, but the exact positions and numbers of binding sites are not clear. Here, we employed a collagen II peptide library to characterize high-affinity binding sites for HSP47. We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. In contrast, large hydrophobic amino acids such as phenylalanine at certain positions in the collagen sequence increase binding strength. For further characterization, we determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large hydrophobic side chain from the middle strand of the collagen triple helix and, most surprisingly, possess an altered binding stoichiometry in the form of a 1:1 complex. This altered stoichiometry is explained by steric collisions with the second HSP47 molecule present in all structures determined thus far caused by the newly introduced large hydrophobic residue placed on the trailing strand. This exemplifies the importance of considering all three sites of homotrimeric collagen as independent interaction surfaces and may provide insight into the formation of higher oligomeric complexes at promiscuous collagen-binding sites.

GET two for one.

In this issue, Voth et al. (2014) reveal that Get3, the GET pathway targeting factor shuttling TA-proteins from the ribosome to the ER membrane, moonlights as a chaperone under oxidizing conditions in a manner reminiscent of bacterial Hsp33.

Modulation of the Hsp90 chaperone cycle by a stringent client protein.

Hsp90 is the most abundant molecular chaperone in the eukaryotic cell. One of the most stringent clients is the glucocorticoid receptor (GR), whose in vivo function strictly depends on the interaction with the Hsp90 machinery. However, the molecular mechanism of this interaction has been elusive. Here we have reconstituted the interaction of Hsp90 with hormone-bound GR using purified components. Our biochemical and structural analyses define the binding site for GR on Hsp90 and reveal that binding of GR modulates the conformational cycle of Hsp90. FRET experiments demonstrate that a partially closed form of the Hsp90 dimer is the preferred conformation for interaction. Consistent with this, the conformational cycle of Hsp90 is decelerated, and its ATPase activity decreases. Hsp90 cochaperones differentially affect formation of the Hsp90-GR complex, serving as control elements for cycle progression and revealing an intricate interplay of client and cochaperones as molecular modulators of the Hsp90 machine.

Regulation of small heat-shock proteins by hetero-oligomer formation.

Small heat-shock proteins (sHsps) compose the most widespread family of molecular chaperones. The human genome encodes 10 different sHsps (HspB1-10). It has been shown that HspB1 (Hsp27), HspB5 (αB-crystallin), and HspB6 (Hsp20) can form hetero-oligomers in vivo However, the impact of hetero-oligomerization on their structure and chaperone mechanism remains enigmatic. Here, we analyzed hetero-oligomer formation in human cells and in vitro using purified proteins. Our results show that the effect of hetero-oligomer formation on the composition of the sHsp ensembles and their chaperone activities depends strongly on the respective sHsps involved. We observed that hetero-oligomer formation between HspB1 and HspB5 leads to an ensemble that is dominated by species larger than the individual homo-oligomers. In contrast, the interaction of dimeric HspB6 with either HspB1 or HspB5 oligomers shifted the ensemble toward smaller oligomers. We noted that the larger HspB1-HspB5 hetero-oligomers are less active and that HspB6 activates HspB5 by dissociation to smaller oligomer complexes. The chaperone activity of HspB1-HspB6 hetero-oligomers, however, was modulated in a substrate-specific manner, presumably due to the specific enrichment of an HspB1-HspB6 heterodimer. These heterodimeric species may allow the tuning of the chaperone properties toward specific substrates. We conclude that sHsp hetero-oligomerization exerts distinct regulatory effects depending on the sHsps involved.

Acidification activates ERp44--a molecular litmus test for protein assembly.

In this issue of Molecular Cell, Vavassori et al. (2013) show that a pH-induced conformational change in the quality control protein ERp44 allows retrieval of secretory proteins that contain free thiols via a disulfide linkage from postendoplasmic reticulum compartments to prevent their premature secretion.