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Hsp90 Chaperones: Tools in Protein Folding

Cells are equipped with several classes of chaperones that ensure proper folding of newly synthesized proteins or stress denatured proteins. Different chaperones follow distinct strategies and cross-talk with each other to achieve the ultimate goal of correcting misfolded polypeptides. For example, a polypeptide bound to Hsp70 at its early stage of folding might be transferred to a chaperonin to reach its final native stage. In case of certain specialized polypeptides in the eukaryotic cytosol, substrate binding to Hsp70 is followed by interaction with a 90 kD chaperone Hsp90 (2,3). Hsp90 is one of the most abundant proteins in eukaryotes, accounting for ~1% of the total soluble protein even under non-stressed conditions (1). Unlike other chaperones, Hsp90 does not act in nascent protein folding. It binds to substrate proteins, which are in a near native state, at a later stage of folding (2,3).

Structure and mode of action

Hsp90 reaction cycle along with co-chaperones hsp90
Domain Organisation of Hsp90

The Hsp90 family is highly conserved. It includes HtpG in the bacterial cytosol, Grp94/gp96 in the endoplasmic reticulum of eukaryotes, Hsp75/TRAP1 in the mitochondrial matrix and Hsp90 in eukaryotic cytosol. Cytosolic Hsp90s are further termed as Hsc82 and Hsp82 in yeast, Hsp83 in Drosophila, Hsp86 and Hsp84 in mice, Hsp90α and Hsp90β in humans. All these homologs share a common structural plan and hence have a similar mode of action (2). Hsp90 forms a constitutive homodimer through its carboxy-terminal residues. The amino-terminal region is highly conserved and contains the ATP binding pocket. N-terminal domain is followed by a conserved and structurally flexible middle domain. The middle domain and the N-terminal domain are separated by a divergent charged sequence. Both N- and C-terminal domain is capable of binding to a substrate (4). Polypeptide binding at the N-terminal domain is the nucleotide dependent and is also affected by the adjacent charged sequence (1). The ATP-binding domain of Hsp90 is structurally related to the superfamily of homodimeric ATPases like DNA gyrase and topoisomerase II. ATP binding results in dimerization of the nucleotide binding domains and result in formation of circular structures (2,4). This interaction between the N-terminal domains is essential for ATP hydrolysis. ATP-bound state of Hsp90 binds stably to substrate polypeptides whereas the substrate release is achieved through ATP hydrolysis, probably by opening up the Hsp90 dimer (1,4). In its ATP clamp state, Hsp90 encompasses a sizable domain of the substrate by optimally exposing the substrate binding face (4).

Interrelationship of Hsp90 and its co-chaperones

Bacterial and endoplasmic reticulum forms of Hsp90 operate independently (7). Hsp90 in the eukaryotic cytosol interacts with a variety of cochaperone proteins that assemble into a multichaperone complex and regulate its function (2, 5, 3). Binding to Hsp90 is largely modulated by co-chaperones containing a modular domain with three 34 amino acid, helix-turn-helix tetratricopeptide repeat (TPR) motifs. Hsp90 binding TPR domains have also been identified fused to other TPR domains, which recognize Hsp70 and then connect Hsp70 to Hsp90 (examples are p60, Hop and Sti1) or recruit Hsp90 to the mitochondrial import machinery (example- Tom34) (7, 2, 4). Recently, a TPR cofactor CHIP has been shown to link Hsp90 with the ubiquitination apparatus, controlling protein degradation by the proteasome (7). The TPR domain cochaperones bind at the COOH terminus of Hsp90 (2, 4). In the case of the cochaperone Hop; the TPR domain recognizes the signature COOH-terminal residues MEEVD of Hsp90 that are anchored by interactions with several conserved residues in the TPR domain that form a “two-carboxylate clamp” (2, 4). Similarly, a separate TPR domain of Hop in a similar fashion with the last eight residues of Hsc70 (GPTIEEVD) held in the groove of the TPR domain and anchored by an identical carboxylate clamp (4). Hop acts as an inhibitor of the Hsp90 ATPase by preventing access to the nucleotide-binding site of Hsp90 and has been proposed to be part of a substrate loading mechanism for Hsp90 (1). Hsp90-Hop-Hsc70 complex permit's transfer of a substrate polypeptide from Hsc70 to the nucleotide-free state of Hsp90. Binding of ATP to Hsp90 displaces the Hop-Hsc70 loading system and simultaneously close the substrate binding clamp of Hsp90 (2).

Physiological functions

Hsp90 is distinguished from other chaperones in that most of its known substrates are signal transduction proteins such as steroid hormone receptors and signaling kinases (2). It maintains the activity of the signaling proteins that play a key role in cellular signal transduction networks. A short description of the role o Hsp90 in maintenance of chromatin structure and cell communication is mentioned below.

Role in signal transduction

Hsp90 chaperone machinery plays a critical role in maturation and conformational maintenance of steroid hormone receptors, signal transducers, several tyrosine and serine/threonine kinases, cell-cycle regulators and disparate proteins such as nitric oxide synthase and calcineurin (6). Disruption of Hsp90 affects multiple stages of the mitogenic signal cascade, cyclin-dependent progression through both G1 and G2, and centrosome function during mitosis (1, 5). These large multidomain proteins require stabilizing interactions with other factors such as Hsp90 for their productive interaction with the ligand (2). Signaling proteins existing in many regulatory states often undergo a conformational switch. The structural flexibility needed for these steps renders them inherently less stable and thus requires stabilizing interactions with Hsp90 (6, 7). For example, interaction of the glucocorticoid receptor with Hsp90 is essential for its activity. The unstable ligand-binding domain of the receptor is sufficient for its interaction with the chaperone. Monomeric glucocorticoid receptor is loaded onto Hsp90 by the Hsp70 chaperone machinery, and it attains its hormone-binding conformation after binding to Hsp90. Once the folded monomeric receptor has been released from the chaperones, it either binds the appropriate steroid hormone resulting in its dimerization and activation or remains unstable and is recognized again by the chaperone machinery (2, 4).

Hsp90 as a capacitor of evolution

Genetic variation is a natural phenomenon that occurs constitutively in a wild population and is potentiated by environment and genetic background. It is one of the prime determinants of molecular evolution. HSP90 plays an integral role in the evolutionary processes. Through its low-affinity, interactions characterized by repeated cycles of binding and release it can conceal inherent genetic variation within a population via its protein chaperoning function (3). Hsp90 acts as a biochemical buffer and stably binds to the mutant substrates and thereby, does not allow their phenotypic expression. This allows accumulation of polymorphic variants of crucial signaling pathways while the pathway as a whole retains sufficient function to maintain wild-type phenotypes (8). Multiple previously silent genetic determinants produced by these variants when enriched by selection, rapidly became independent of the Hsp90 buffering mechanism. This results in expression of the cryptic variants, and further positive selection leads to the continued expression of these traits (8, 9).

Role in chromosome maintenance

Studies from Drosophila melanogaster have shown that compromised HSP90 function induces epigenetic alterations in gene expression as well as heritable alterations in the chromatin state. HSP90 co-factors interact with the DNA helicases that form key components of general chromatin-remodeling complexes (5). Recently, it has been shown that Hsp90 influence telomere protein biology and interacts with a subunit human telomerase hTERT. They promote DNA binding and nucleotide affinity for telomerase, and hence contribute to telomere DNA length maintenance. In cancer, telomerase upregulation reduces replicative senescence and causes the cell to perpetuate. This increased telomerase activity was found to co-relate with a rise in HSP90 (10). Modulation of telomerase activity through HSP90 dependent mechanisms occur in numerous cancer types, including hepatomas, adrenalomas, glioblastomas and melanomas.

Hsp90 and cancer

HSP90 functions as a biochemical buffer of the extensive genetic heterogeneity that is found in most of the cancers (8). However, this buffering mechanism may break down upon an increase in load of mutant and misfolded oncoproteins. As a result, the phenotypic diversity within the tumour cell population would increase and accelerate the development of invasive, metastatic and drug resistant cells (3). Hsp90 and its co-factors also modulate apoptosis of tumour cells. It inhibits apoptosis via its interaction with AKT, TNF receptor and NF-κB. Hsp90 is involved in dynamic low affinity interactions with the transcription factors and kinases and maintain them in a latent but readily activated state. Oncogenic mutations in such proteins cause them to be in unstable conformation and demand higher expression of the chaperone (7, 11). For example, SRC tyrosine kinase has a catalytic subunit and a regulatory subunit. The regulatory subunit represses the kinase activity of SRC and stabilizes its structure by interacting with the SH2 domain of the protein. Mutations truncating the regulatory region of SRC results in a constitutively active but conformationally unstable kinase. Truncated SRC associates much stably with Hsp90 as compared to wild type. This aberrant chaperone interaction causes acquisition and maintenance of the increased kinase activity of mutant SRC, underlying its transforming activity. Similarly, mutations in tumour suppressor protein p53 are the most common molecular genetic defect found in human cancers (11). Most p53 mutations result in altered protein conformation and impaired cell-cycle checkpoint activity. Wild-type p53 protein is short-lived. HSP90 machinery maintains it in a state that is activation competent and regulates its degradation through the ubiquitin-proteasome system (7). Mutant p53 proteins due to their aberrant conformation display extended binding to the chaperone and hence prevent their normal ubiquitylation and degradation. Subsequent accumulation of dysfunctional protein may be a dominant negative effect on tumour suppressive effect of wild-type proteins and trans-activate other oncocytic pathways (11). The high prevalence of HSP90 in a broad range of cancers makes it an ideal candidate for molecular target in anticancer therapeutics.


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Database Statistics

Total Number of Genomes
Numbers in Bacteria
Numbers in Archaea
Numbers in Protists
Numbers in Fungi
Numbers in Algae
Numbers in Plants
Numbers in Animals
Total Number of Hsp90 Proteins