Hsp90: Heat Shock Protein 90 (PDB ID = 2CG9)
Created by Vincent Nguyen-Cao
Hsp90 is a heat shock protein that acts as a chaperone to other proteins during extreme heat cellular conditions (7). This important protein serves to maintain the active form of many eukaryotic signaling proteins during stress by assisting in protein folding, cell signaling, protein homeostasis and tumor repression. This particular hsp90 is found in Saccharomyces cerevisiae, or baker’s yeast (pdb id=2CG9). Hsp90 is a dimer that exists in its inactive (opened) and active (closed) conformation. These conformations are regulated by a complex mechanism of ATPase-coupled conformational changes (1)(7). Furthermore, Hsp90 has an isoelectric point of 5.12 and a molecular weight of 187383.22, or roughly 90 kDa (6). Its structural diversity permits it to expand a wide range of clientele proteins that include: a variety of transcription factors, protein kinases, cell cycle regulators, and steroid receptors (14)(7).
Due to Hsp90’s ubiquitous nature in serving as a molecular chaperone, it has become a focus in chemotherapy. As biochemical buffers, chaperone proteins like Hsp90 facilitate the tolerance of mutated signaling molecules that would otherwise be deemed lethal to the cell (2).These malignant transformations are potentially oncogenic. Though Hsp90 is essential in normal cellular activity, cancerous cells are more dependent on Hsp90 than normal cells (7). So, drugs that block Hsp90 function, for example, by blocking the binding of ATP ligand, may facilitate the accumulation of misfolded proteins (17). This buildup stimulates the proteosome system to destroy these proteins, corrupting the signaling pathways and effectively eliminating the potential cancer cell growth (7). By understanding Hsp90’s role and relationships with its clientele proteins, insight is gained into how its structure, properties, and function may be utilized as an integral tool for innovative cancer therapy.
The 90-kDa heat shock protein is an obligate dimer composed of three domains per monomer. ATP binds to the N-terminal domain, the middle domain is suggested to be involved in client binding, and the C-terminal domain is the site of dimerization (9). Hsp90 is composed of α-helices, β-sheets, and random coils. Due to the cytoplasmic environment where Hsp90 is commonly found, it is largely globular in structure with nonpolar hydrophobic residues on the inside and polar residues on the outside.
The N-terminal domain is about 25 kDa and is composed of a two-layer α/β sandwich structure, where the alpha helices create a pocket that extends from the surface to the buried face of the highly twisted antiparallel β-sheet (14). This pocket is the ATP/ADP-binding site. Key residue sites in the N-terminal domain revealed that the biological function of Hsp90 is dependent on its ability to bind and hydrolyze ATP (14). These key residues in the yeast Hsp90 include: Asp-79, which forms hydrogen bonds with the adenine base of ATP, and Glu-33, which is involved ATPase activity and ATP binding. Moreover, magnesium ions are required for ATP binding to Hsp90; however, research indicates that the N-domain has no inherent Mg2+ binding sites (17).
This N-terminal binding pocket has been the focus of the antitumor drug, geldanamycin, which is found in Streptomyces bacteria (pdb id=1yet). Geldanamycin was found to bind to the ATP-binding site on Hsp90 via high density van der Waals interactions (17). The key residue for this binding between the Hsp90 interface and geldanamycin was determined to be the Asp-93 residue found at the bottom of the hydrophobic pocket. As this drug binds to Hsp90, it successfully blocks the ATP-induced conformational changes needed for normal chaperone function (14). Therefore, geldanamycin is a highly specific competitive inhibitor. Additionally, this discovery successfully confirmed and identified the N-terminal pocket as the binding site for adenine nucleotides of ATP. Paired with Hsp90’s correlation with cancer cells, geldanamycin derivatives are regarded as antitumor agents. Ultimately, geldanamycin was found to be too toxic for pharmaceutical use; herbimycin and macbecin are closely related analogs that are used as antitumor antibiotics (17). Other promising ATP-competitive inhibitors include 17-AAG, 17-DMAG, BBII0121 and NVP-AUY922 (14).
The middle segment of Hsp90 is about 35 kDa and consists of three subdomains. The large αβα domain (first subdomain) at the N terminus is connected via a short α3 helical coil (second subdomain) to a small αβα domain (third subdomain) at the C-terminus (14). Research has shown DNA gyrase B shares an ATP-binding sequence homology to Hsp90; this relationship between structure and function may explain how Hsp90’s larger αβα is equivalent to a αβα motif in DNA gyrase B (pdb=1EI1). Overall, the middle segment of the Hsp90 is a major site for client protein interactions. This can be attributed to the conserved hydrophobic patch centered on Trp-300 and the unique amphipathic protrusion formed by residues 327-340 that play a role in client protein binding (14). Other protein complexes, like the Hsp70-Hsp40 machinery or the adaptor protein HOP can contribute to this client protein binding. Additionally, Arg380 is found in a short α-helix catalytic loop within this domain and plays an important role in polarizing the β-γ-phosphodiester bond of ATP (14).
The last domain, the 10kDa C-terminal domain, is responsible for Hsp90 dimerization. This domain is formed by a mixture of α-helices and β-sheets. Residues 587-610 are involved in dimeric interactions with the equivalent protomer. The C-terminal end of this domain consists of a 4 α-helices bundle. New research suggests the possibility that the carboxyl-terminus also has an ATP-binding domain. This would imply that both ends of Hsp90, the amino- and carboxyl- terminal domains, interact and modulate chaperone activity (10). It should be noted that in this particular yeast C-domain, the highly conserved EEVD amino acid sequence motif that typically binds to co-chaperones with tetratricopeptide repeat (TPR) domains, is disordered (14).
Hsp90 is regarded to either exist as an inactive, V-shape, open conformation (pdb=2IOQ), or an ATP-bound, closed conformation (pdb=2CG9). Thus, the ATPase cycle is coupled to the opening and closing of a molecular clamp. The open conformation is a linear antiparallel arrangement of two monomers dimerized via the C-terminus with a left-handed helical twist. The closed conformation mechanism, on the other hand, involves N-terminal dimerization with a high affinity for ATP-binding (14). This N-terminal dimerization is essential for ATP hydrolysis and brings the middle domains of the dimer 20 Å closer together (1).
Here, the critical step in Hsp90 conformation change is the movement of the “lid segment” (residues 94-125) rotating 180° from the open conformation to the closed conformation. The“lid” swings from the “hinges” of Gly95 and Gly121 to fold over the previously described nucleotide-binding pocket of the N-terminal domain (1). The movement of this lid reveals the hydrophobic patch of the middle domain which will interact, via hydrogen bonds, with the middle domain counterpart of the opposite monomer. Again, this hydrophobic patch stabilizes the N-domain dimerization in the closed conformation (1). Additionally, this ATP-bound conformation is regarded as “tense” and is dependent on the γ-phosphate of ATP to stabilize it. The γ-phosphate is supported by the glycine-rich loop of the C-terminal hinge and forms hydrogen bonds with Gln-119, Gly-121, Val-122, and Gly-123 (1). The β-phosphate forms hydrogen bonds with the peptide backbone at Phe-124 and Gly-100 from the N-terminal hinge (1). Next, the β-γ phosphodiesterbond is attacked by a water molecule activated by Glu-33—the essential catalytic site of the N-terminal domain (1). Arg-380, another key residue site, polarizes the β-γ-phosphodiester bond to neutralize the transition state (1). Subsequent hydrolysis cleaves the ATP to ADP and destabilizes the closed conformation—allowing the dimer to relax to the open, inactive state (14). This ATPase coupled conformational cycle is likely to be conserved in human hsp90 equivalents.
In summary, a client protein is able to bind, in the absence of ATP, to the open conformation (pdb=2IOQ). The client protein adopts a near-native conformation to bind appropriately with respect to Hsp90 (via the rotation of the hinge residues). This triggers the binding of ATP. The binding of ATP creates the closed conformation (pdb=2CG09) by inducing N-terminal dimerization, which is stabilized by co-chaperones. During this “tense” phase, the client protein maybe modified by the Hsp90 chaperone properties. ATP is then cleaved into ADP and Pi (triggered by the co-chaperone Aha1) and Hsp90 returns to the open state once the client protein is released (3). Furthermore, this ATP-binding site of the N-terminus domain is the focus of intense research because inhibition by drugs like, geldanamycin, blocks client prote inactivation—which may prove to be key in limiting the growth of cancerous cells. Literature suggests the rate of chaperone activity is not dependent on ATP hydrolysis, but rather, the ability of the catalytic N-domain and the catalytic loop of the middle domain to close the “lid” of the N-domain and position the complex for ATP hydrolysis (1).
This particular crystallization of yeast Hsp90 (pdb=2CG9) is in complex with the co-chaperone p23 (more specifically, its yeast homologue Sb1). P23/Sb1 has been found to have a late role role in the chaperone cylcle by being able to enhance the release of the active client protein (14). This co-chaperone also has an inhibitory influence (up to 50%) on the ATPase activity. This decline in Hsp90 activity is attributed to the observation that p23/Sba1 binds to the ATP-bound closed conformation of Hsp90 and is therefore committed to ATP hydrolysis (14). This co-chaperone interacts with Hsp90 at three interfaces and stabilizes the ATP-bound closed conformation.
Other co-chaperones, like p23/Sba1, function to regulate the chaperone ATPase cycle by interacting with the switch components of the N-terminal hinge during the ATP-bound closed conformation. Aha1 enables the catalytic loop to come into contact with ATP by lowering the energy barrier to increase chaperone activity (1). On the other hand, the Cdc37 co-chaperone locks the Hsp90 lid segment in place at the open conformation, prevents N-terminal dimerization, engages the catalytic residue Glu-33 in a polar interaction, and blocks access to the catalytic loop containing Arg-380. The combination of these processes successfully blocks the structural rearrangements needed to bind an ATP molecule. As a result, subsequent Hsp90 chaperone folding processes on clientele proteins will not be possible. (1).
Hsp90 belongs to a class of heat shock chaperone proteins and shares structural similarities with proteins that act in a similar manner. One such protein, GRP94-nucleotide complex (pdb=2O1U), is regarded as the essential endoplasmic reticulum chaperone paralog of hsp90. GRP94 is found in Canis lupus familiaris (dogs). Following BLAST results, GRP94 was found to have an E-score of 2×10-163, which indicates very strong amino acid sequence similarity and conserved homology (4). By amino acid homology, GRP94 is about 60% homologous and 50% identical to Hsp90 (12). This chaperone protein is involved in the conformational maturation of other proteins programmed to cell-surface display or cell export. GRP94 client proteins are often secretory proteins, membrane proteins, and proteins involved in proper immune responses (13). Unlike Hsp90, GRP94 is only weakly expressed under heat shock conditions; its expression is more stimulated under animal ischemic injuries and cellular stress (13).
Hsp90 shares tertiary structural similarity with GRP94. DALI results show a Z-score of 21.8 and a rmsd of 3.1 Å (from the backbone of Hsp90)—indicating a strong GRP94 three-dimensional structure correlation to Hsp90 (8). GRP94 activity is not regulated by co-chaperones or accessory proteins (13), unlike Hsp90 which typically requires co-chaperones for it to function properly. Recent research has also shown that GRP94 is linked to potential cancer therapies (5). Like Hsp90, the N-terminal domains of Grp94 contains conserved ATP/ADP binding sites, however, ATP binding alone does not drive GRP94 into a productive conformation. This suggests a possible mechanistic divergence from Hsp90 to cater to a specific set of client proteins (13). Literature indicates the alignment of the ATP catalytic residues differ between the N and M domains of Hsp90 and GRP94 (5). The GRP94 dimer instead adopts a relaxed “twisted V” conformation that prevents the proper alignment of the Glu-33 and Arg-380 residues that is typically stabilized by the N domain dimer, the bound ATP, and a co-chaperone in Hsp90 (5).
Hsp90 is an important chaperone protein that primarily aids in protein synthesis by binding to clientele protein during extreme heat cellular conditions. It exists as a dimer that is directed, by the binding of ATP, to take on an active or inactive state. Consisting of three domains, Hsp90 is composed of a variety of key residues that aid in the binding of ATP and subsequent chaperone capabilities (for example, protein unfolding). Along with ATP, co-chaperones may bind to Hsp90 to stimulate or repress chaperone activity. Undoubtedly, Hsp90 is a significant protein whose structure determines its function. With that said, this protein shares many primary, secondary, and tertiary structure similarities with related proteins. The ATP binding site in the N-terminal domain is of particular interest in drug development research. Due to Hsp90’s strong presence in cancer cells, blocking the chaperone activity of Hsp90 may be a potential cancer cell treatment. Thus, Hsp90 is currently designated as a protein chaperone with considerable biological focus.