Structural dynamics of RAF1-HSP90-CDC37 and HSP90 complexes reveal asymmetric client interactions and key structural elements.

RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 µs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1's unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.

Rational design of peptide inhibitors targeting HSP90-CDC37 protein-protein interaction.

Background: Specifically blocking HSP90-CDC37 interaction is emerging as a prospective strategy for cancer therapy. Aim: Applying a kinase pseudopeptide rationale to the discovery of HSP90-CDC37 protein-protein interaction (PPI) inhibitors. Methods: Pseudosubstrates were identified through sequence alignment and evaluated by biolayer interferometry assay, co-immunoprecipitation assay and antiproliferation assay. Results: TAT-DDO-59120 was identified to disrupt HSP90-CDC37 PPI through directly binding to HSP90, both extracellularly and intracellularly. In addition, the identified peptide showed ideal antiproliferative activity against the colorectal cancer cell HCT116 (IC50 = 12.82 µM). Conclusion: Compared with the traditional method of screening a large compound library to identify PPI inhibitors, this method is rapid and efficient with strong purpose, which provides a novel strategy for designing HSP90-CDC37 PPI inhibitors.

Crystal structures of dimeric and heptameric mtHsp60 reveal the mechanism of chaperonin inactivation.

Mitochondrial Hsp60 (mtHsp60) plays a crucial role in maintaining the proper folding of proteins in the mitochondria. mtHsp60 self-assembles into a ring-shaped heptamer, which can further form a double-ring tetradecamer in the presence of ATP and mtHsp10. However, mtHsp60 tends to dissociate in vitro, unlike its prokaryotic homologue, GroEL. The molecular structure of dissociated mtHsp60 and the mechanism behind its dissociation remain unclear. In this study, we demonstrated that Epinephelus coioides mtHsp60 (EcHsp60) can form a dimeric structure with inactive ATPase activity. The crystal structure of this dimer reveals symmetrical subunit interactions and a rearranged equatorial domain. The α4 helix of each subunit extends and interacts with its adjacent subunit, leading to the disruption of the ATP-binding pocket. Furthermore, an RLK motif in the apical domain contributes to stabilizing the dimeric complex. These structural and biochemical findings provide new insights into the conformational transitions and functional regulation of this ancient chaperonin.

A molecular device for the redox quality control of GroEL/ES substrates.

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.

Cdc37 as a Co-chaperone to Hsp90.

The co-chaperone p50/Cdc37 is an important partner for Hsp90, assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Analysis of the structure of Hsp90-Cdc37-kinase complexes demonstrates the way in which Cdc37 interacts with and controls the folding of a large proportion of intracellular protein kinases. This co-chaperone thus stands at the hub of a multitude of intracellular signaling networks. Indeed, the influence of Cdc37 reaches beyond the housekeeping pathways of protein folding into the regulation of a wide range of cellular processes. This co-chaperone has attracted attention as a potential intermediate in carcinogenesis. Cdc37 is an attractive potential target in cancer due to (1) high expression in a number of tumor types and (2) control of multiple signaling pathways. These properties indicate (3) a potential for selectivity due to its elevated expression in malignant cells and (4) robustness, as the co-chaperone may control multiple growth signaling pathways and thus be less prone to evolution of resistance than less versatile oncoproteins. Cdc37 may also be involved in other aspects of pathophysiology and has been shown to be secreted in exosomes. Protein aggregation disorders have been linked to age-related declines in molecular chaperones and co-chaperones. Cdc37 also appears to be a potential agent in longevity due to its links to protein folding and autophagy, and it will be informative to study the role of Cdc37 maintenance/decline in aging organisms.

Chaperonin: Co-chaperonin Interactions.

Co-chaperonins function together with chaperonins to mediate ATP-dependent protein folding in a variety of cellular compartments. Chaperonins are evolutionarily conserved and form two distinct classes, namely, group I and group II chaperonins. GroEL and its co-chaperonin GroES form part of group I and are the archetypal members of this family of protein folding machines. The unique mechanism used by GroEL and GroES to drive protein folding is embedded in the complex architecture of double-ringed complexes, forming two central chambers that undergo conformational rearrangements that enable protein folding to occur. GroES forms a lid over the chamber and in doing so dislodges bound substrate into the chamber, thereby allowing non-native proteins to fold in isolation. GroES also modulates allosteric transitions of GroEL. Group II chaperonins are functionally similar to group I chaperonins but differ in structure and do not require a co-chaperonin. A significant number of bacteria and eukaryotes house multiple chaperonin and co-chaperonin proteins, many of which have acquired additional intracellular and extracellular biological functions. In some instances, co-chaperonins display contrasting functions to those of chaperonins. Human HSP60 (HSPD) continues to play a key role in the pathogenesis of many human diseases, in particular autoimmune diseases and cancer. A greater understanding of the fascinating roles of both intracellular and extracellular Hsp10 on cellular processes will accelerate the development of techniques to treat diseases associated with the chaperonin family.

Mechanistic insights into protein folding by the eukaryotic chaperonin complex CCT.

The cytosolic chaperonin CCT is indispensable to eukaryotic life, folding the cytoskeletal proteins actin and tubulin along with an estimated 10% of the remaining proteome. However, it also participates in human diseases such as cancer and viral infections, rendering it valuable as a potential therapeutic target. CCT consists of two stacked rings, each comprised of eight homologous but distinct subunits, that assists the folding of a remarkable substrate clientele that exhibits both broad diversity and specificity. Much of the work in recent years has been aimed at understanding the mechanisms of CCT substrate recognition and folding. These studies have revealed new binding sites and mechanisms by which CCT uses its distinctive subunit arrangement to fold structurally unrelated substrates. Here, we review recent structural insights into CCT-substrate interactions and place them into the broader context of CCT function and its implications for human health.

Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.

Structural and Functional Features of Viral Chaperonins.

Chaperonins provide proper folding of proteins in vivo and in vitro and, as was thought until recently, are characteristic of prokaryotes, eukaryotes, and archaea. However, it turned out that some bacteria viruses (bacteriophages) encode their own chaperonins. This review presents results of the investigations of the first representatives of this new chaperonin group: the double-ring EL chaperonin and the single-ring OBP and AR9 chaperonins. Biochemical properties and structure of the phage chaperonins were compared within the group and with other known group I and group II chaperonins.

Snapshots of actin and tubulin folding inside the TRiC chaperonin.

The integrity of a cell's proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.

Chaperonin Mechanisms: Multiple and (Mis)Understood?

The chaperonins are ubiquitous and essential nanomachines that assist in protein folding in an ATP-driven manner. They consist of two back-to-back stacked oligomeric rings with cavities in which protein (un)folding can take place in a shielding environment. This review focuses on GroEL from Escherichia coli and the eukaryotic chaperonin-containing t-complex polypeptide 1, which differ considerably in their reaction mechanisms despite sharing a similar overall architecture. Although chaperonins feature in many current biochemistry textbooks after being studied intensively for more than three decades, key aspects of their reaction mechanisms remain under debate and are discussed in this review. In particular, it is unclear whether a universal reaction mechanism operates for all substrates and whether it is passive, i.e., aggregation is prevented but the folding pathway is unaltered, or active. It is also unclear how chaperonin clients are distinguished from nonclients and what are the precise roles of the cofactors with which chaperonins interact.

In silico identification of small molecule modulators for disruption of Hsp90-Cdc37 protein-protein interaction interface for cancer therapeutic application.

The protein-protein interactions (PPIs) in the biological systems are important to maintain a number of cellular processes. Several disorders including cancer may be developed due to dysfunction in the assembly of PPI networks. Hence, targeting intracellular PPIs can be considered as a crucial drug target for cancer therapy. Among the enormous and diverse group of cancer-enabling PPIs, the Hsp90-Cdc37 is prominent for cancer therapeutic development. The successful inhibition of Hsp90-Cdc37 PPI interface can be an important therapeutic option for cancer management. In the current study, a set of more than sixty thousand compounds belong to four databases were screened through a multi-steps molecular docking study in Glide against the Hsp90-Cdc37 interaction interface. The Glide-score and Prime-MM-GBSA based binding free energy of DCZ3112, standard Hsp90-Cdc37 inhibitor were found to be -6.96 and -40.46 kcal/mol, respectively. The above two parameters were used as cut-off score to reduce the chemical space from all successfully docked molecules. Furthermore, the in-silico pharmacokinetics parameters, common-feature pharmacophore analyses and the molecular binding interactions were used to wipe out the inactive molecules. Finally, four molecules were found to be important to modulate the Hsp90-Cdc37 interface. The potentiality of the final four molecules was checked through several drug-likeness characteristics. The molecular dynamics (MD) simulation study explained that all four molecules retained inside the interface of Hsp90-Cdc37. The binding free energy of each molecule obtained from the MD simulation trajectory was clearly explained the strong affection towards the Hsp90-Cdc37. Hence, the proposed molecule might be crucial for successful inhibition of the Hsp90-Cdc37 interface.Communicated by Ramaswamy H. Sarma.

Okicamelliaside targets the N-terminal chaperone pocket of HSP90 disrupts the chaperone protein interaction of HSP90-CDC37 and exerts antitumor activity.

Heat shock protein 90 (HSP90) has been recognized as a crucial target in cancer cells. However, various toxic reactions targeting the ATP binding site of HSP90 may not be the best choice for HSP90 inhibitors. In this paper, an ellagic acid derivative, namely, okicamelliaside (OCS), with antitumor effects was found. To identify potential anti-cancer mechanisms, an OCS photosensitive probe was applied to target fishing and tracing. Chemical proteomics and protein-drug interaction experiments have shown that HSP90 is a key target for OCS, with a strong binding affinity (KD = 6.45 µM). Mutation analysis of the target protein and molecular dynamics simulation revealed that OCS could competitively act on the key Glu-47 site at the N-terminal chaperone pocket of HSP90, where the co-chaperone CDC37 binds to HSP90, affect its stability and reduce the ∆Gbind of HSP90-CDC37. It was demonstrated that OCS destroys the protein-protein interactions of HSP90-CDC37; selectively affects downstream kinase client proteins of HSP90, including CDK4, P-AKT473, and P-ERK1/2; and exerts antitumor effects on A549 cells. Furthermore, tumor xenograft experiments demonstrated high antitumor activity and low toxicity of OCS in the same way. Our findings identified a novel N-terminal chaperone pocket natural inhibitor of HSP90, that is, OCS, which selectively inhibits the formation of the HSP90-CDC37 protein complex, and provided further insight into HSP90 inhibitors for anti-cancer candidate drugs.

Protein interaction landscapes revealed by advanced in vivo cross-linking-mass spectrometry.

Defining protein-protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking-mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry-based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine-lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns.

The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

HSP-90/kinase complexes are stabilized by the large PPIase FKB-6.

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.

Discovery of a covalent inhibitor of heat shock protein 90 with antitumor activity that blocks the co-chaperone binding via C-terminal modification.

Heat shock protein (Hsp90), a critical molecular chaperone that regulates the maturation of a large number of oncogenic client proteins, plays an essential role in the growth of neoplastic cells. Herein, DDO-6600 is identified to covalent modification of Cys598 on Hsp90 from in silico study and is verified by a series of biological assays. We demonstrated that DDO-6600 covalently bound to Cys598 on the Hsp90 C terminus and exhibited antiproliferative activities against multiple tumor cells without inhibiting ATPase activity. Further studies showed that DDO-6600 disrupted the interaction between Hsp90 and Cdc37, which induced the degradation of kinase client proteins in multiple tumor cell lines, promoted apoptosis, and inhibited cell motility. Our findings offer mechanic insights into the covalent modification of Hsp90 and provide an alternative strategy for the development of Hsp90 covalent regulators or chemical probes to explore the therapeutical potential of Hsp90.

Chaperonin point mutation enhances cadmium endurance in Saccharomyces cerevisiae.

OBJECTIVE: To study the effect of the mutation in conserved G412E in Cct7p subunit of CCT complex on its cellular fate. RESULTS: TriC/CCT is a dynamic multimeric protein that assists in protein folding in an energy-dependent manner. A point mutation in the ATP binding pocket in the equatorial domain of the Cct7p subunit delays the doubling time. The cell size was twice the wild type, and the formation of protein aggregates suggests disturbed folding of the proteins. Upon growing in stressful conditions of arsenous acid and cadmium chloride, the mutant was lethal in As3+ but grew well in Cd2+ with 10.5 µg cadmium uptake mg-1 compared to the wild type. The increased expression of vacuole transporters YCF1 and BPT1 by ten-fold and two-fold in mutant indicates the metal transportation to the vacuole. CONCLUSION: CCT complex was vulnerable to the mutation in G412E in the Cct7p subunit of protein folding molecular machinery. Interestingly, already stressed cells provided robustness against oxidative stress and cadmium sequestration in the vacuole.

TEM and STEM-EDS evaluation of metal nanoparticle encapsulation in GroEL/GroES complexes according to the reaction mechanism of chaperonin.

Escherichia coli chaperonin GroEL, which is a large cylindrical protein complex comprising two heptameric rings with cavities of 4.5 nm each in the center, assists in intracellular protein folding with the aid of GroES and adenosine triphosphate (ATP). Here, we investigated the possibility that GroEL can also encapsulate metal nanoparticles (NPs) up to ∼5 nm in diameter into the cavities with the aid of GroES and ATP. The slow ATP-hydrolyzing GroELD52A/D398A mutant, which forms extremely stable complexes with GroES (half-time of ∼6 days), made it possible to analyze GroEL/GroES complexes containing metal NPs. Scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy analysis proved distinctly that FePt NPs and Au NPs were encapsulated in the GroEL/GroES complexes. Dynamic light scattering measurements showed that the NPs in the GroEL/GroES complex were able to maintain their dispersibility in solution. We previously described that the incubation of GroEL and GroES in the presence of ATP·BeFx and adenosine diphosphate·BeFx resulted in the formation of symmetric football-shaped and asymmetric bullet-shaped complexes, respectively. Based on this knowledge, we successfully constructed the football-shaped complex in which two compartments were occupied by Pt or Au NPs (first compartment) and FePt NPs (second compartment). This study showed that metal NPs were sequentially encapsulated according to the GroEL reaction in a step-by-step manner. In light of these results, chaperonin can be used as a tool for handling nanomaterials.

Measuring protein stability in the GroEL chaperonin cage reveals massive destabilization.

The thermodynamics of protein folding in bulk solution have been thoroughly investigated for decades. By contrast, measurements of protein substrate stability inside the GroEL/ES chaperonin cage have not been reported. Such measurements require stable encapsulation, that is no escape of the substrate into bulk solution during experiments, and a way to perturb protein stability without affecting the chaperonin system itself. Here, by establishing such conditions, we show that protein stability in the chaperonin cage is reduced dramatically by more than 5 kcal mol-1 compared to that in bulk solution. Given that steric confinement alone is stabilizing, our results indicate that hydrophobic and/or electrostatic effects in the cavity are strongly destabilizing. Our findings are consistent with the iterative annealing mechanism of action proposed for the chaperonin GroEL.

All cells contain molecules known as proteins that perform many essential roles. Proteins are made of chains of building blocks called amino acids that fold to form the proteins' three-dimensional structures. Many proteins fold spontaneously into their well-defined and correct structures. However, some proteins fold incorrectly, which prevents them from working properly, and can lead to formation of aggregates that may harm the cell. To prevent such damage, cells have evolved proteins known as molecular chaperones that assist in the folding of other proteins. For example, a molecular chaperone called GroEL is found in a bacterium known as Escherichia coli. This molecular chaperone contains a cavity which prevents target proteins from forming clumps by keeping them away from other proteins. However, it remained unclear precisely how GroEL works and whether enclosing target proteins in its cavity has other effects. Moritella profunda is a bacterium that thrives in cold environments and, as a result, many of its proteins are unstable at room temperature and tend to unfold or fold incorrectly. To study how GroEL works, Korobko et al. used a protein from M. profunda called dihydrofolate reductase as a target protein for the chaperone. A clever trick was then used to determine the folding state of dihydrofolate reductase when inside the chaperone cavity. The experiments revealed that the environment within the cavity of GroEL strongly favors dihydrofolate reductase adopting its unfolded state instead of its folded state. This suggests that GroEL helps dihydrofolate reductase and other incorrectly folded target proteins to unfold, thus providing the proteins another opportunity to fold again correctly. Parkinson's disease, Alzheimer's disease and many other diseases are caused by proteins folding incorrectly and forming aggregates. A better understanding of how proteins fold may, therefore, assist in developing new therapies for such diseases. These findings may also help biotechnology researchers develop methods for producing difficult-to-fold proteins on a large scale.