Hsp90 is an important chaperone that interacts with and refolds its client proteins in a cycle that is driven by the binding and hydrolysis of ATP [3
]. Through the course of its catalytic cycle, Hsp90 undergoes considerable structural changes, and this dynamic nature of Hsp90 is the key in its ability to function as a chaperone [4
]. Hsp90 is in a state of conformational flux, whose overall structure is constantly altered by the binding of various ligands, including ATP/ADP, and co-chaperones (i.e., HOP, Cdc37, p23, Aha1 and immunophilins) [7
]. These ligands bind to specific sites on Hsp90 and alter the conformational equilibrium between the two extreme ‘open’ (apo) and ‘closed’ (ATP-bound) states at any given moment [4
The ATPase activity of Hsp90 is linked to its conformational state, which for eukaryotic Hsp90 is influenced by > 20 co-chaperones, as well as by the binding of client proteins, which serve to drive it through its catalytic cycle [3
]. A functional chaperone cycle was first proposed for eukaryotic Hsp90 based on interaction with steroid hormone receptors [8
] and is a process that is probably conserved among eukaryotic Hsp90 species [9
Association of Hsp90 with its client proteins is believed to be initiated by a priori
interaction with Hsp70 (). The client is presented to Hsp70 by its activator, Hsp40, and binds to it in an ATP-dependent manner. Hsp70 interacting protein then binds to and stabilizes this complex. The dimeric co-chaperone HOP (Sti1 in yeast) binds the Hsp40–Hsp70–client complex to Hsp90, thereby forming an Hsp70–HOP–Hsp90 complex [10
]. HOP interacts with the C terminus of Hsp90 through its tetratricopeptide repeat (TPR) domain as well as to additional sites in the middle domain (MD). Co-chaperones and immunophilins bind to the Hsp70–HOP–Hsp90 complex and facilitate the transfer of client from Hsp70 to Hsp90 to form the intermediate complex. On ATP binding, Hsp90 forms a mature complex containing p23 (Sba1 in yeast) and other co-chaperones such as Cdc37 and immunophilins that catalyze the conformational maturation of the client. The co-chaperone p23 as well as the immunophilins FKBP51, FKBP52 and Cyp-40 displace HOP and Hsp70 leading to the mature complex [11
]. Large conformational changes that occur to Hsp90 subsequent to ATP binding are probably transduced to the client leading to its activation (described below). Following release of the mature client, presumably, Hsp90 can re-enter the cycle and bind another client protein.
A simplified cartoon describing the ATPase cycle of Hsp90
The first X-ray crystal structures, along with electron microscopy (EM) and small-angle X-ray scattering (SAXS) data, obtained for full length bacteria (nucleotide free; AMP-PNP-bound; ADP-bound) [12
] and yeast (AMP-PNP- and Sba1-bound) [13
] Hsp90 as well as mammalian (AMP-PNP; ADP-bound) [14
] Grp94 (the endoplasmic reticulum paralog of cytosolic Hsp90) were critical in revealing particular conformations adopted when bound to specific ligand(s). These structures show that the global architecture is conserved across species and that Hsp90 exists as a homodimeric structure in which individual monomers are characterized by three domains; an N-terminal nucleotide binding domain (NBD), site of ATP binding; the MD, site of co-chaperone and client protein binding and involved in ATP hydrolysis; and a C-terminal dimerization domain (CDD), site of dimerization. The NBD is followed by a linker region which connects it to the MD.
Structural and biochemical studies had shown that Hsp90 function was dependent on the binding and hydrolysis of ATP [15
] and suggested that hydrolysis occurs via a ‘molecular clamp’ mechanism involving dimerization of the NBD in the ATP-bound state [17
]. The crystal structures of Hsp90, together with EM and SAXS data, confirmed the ATPase-coupled molecular clamp mechanism and provided further insight connecting Hsp90 complex structure and conformation to the ATPase cycle. In the absence of bound nucleotide, Hsp90 exists in an ‘open’ conformation. While the precise details linking the ATPase cycle to conformational state have not been entirely elucidated, it is known that dramatic conformational changes occur subsequent to ATP binding, whereby the N-terminal domains closely associate with one another resulting in a ‘closed’ conformation that is capable of hydrolyzing ATP [17
]. EM revealed a distinct ‘compact’ conformation when ADP-bound [12
] and in the absence of any bound ligand, the dimer moves to an ‘open’ state. These structures, however, only present a static picture of Hsp90 at its conformational extremes. In order to examine other conformations between these extremes, more dynamic methods must be used.
The solution structure of Escherichia coli
Hsp90 (HtpG) determined using SAXS [6
] shows some important differences compared to the crystal structure. The apo-conformation in solution is more extended with a wider angle implying that it can accommodate more diverse client proteins. Also, the NBD and the MD are rotated by 40° compared to the crystal structure. This may especially impact the ability of nucleotide binding as Gln122 and Phe123 within the active site lid (residues 100 – 126) are positioned to block nucleotide binding in the apo-conformation. Nucleotide binding requires that the lid region be reorganized and the solution structure is better able to accommodate the necessary structural changes essential for nucleotide binding.
The solution structure of eukaryotic Hsp90 has also been determined using SAXS as well as cryo-EM [19
]. Interestingly, these studies showed that Hsp90 can exist in two open conformations, ‘fully-open’ and ‘semi-open’, and revealed an intrinsic flexibility of Hsp90 that is capable of partial closure of the N-terminal domains even in the absence of a nucleotide.
In an attempt to further tease out the conformational cycle of Hsp90 during ATP binding and hydrolysis, Hessling et al.
] used fluorescence energy resonance transfer (FRET) to propose a model consisting of three distinct conformations between the open and closed conformations. In this model, apo-Hsp90 (open conformation) binds ATP in a rapid manner to yield an ATP-bound conformation, followed by the slow formation of an intermediate (I1
) in which the N-terminal domains remain undimerized. While it is not known with certainty, I1
may represent an intermediate in which the ATP lid is closed and the segment on the N-terminal domain required for dimerization is exposed. Subsequent dimerization of the N-terminal domains yields another intermediate (I2
). Next, rearrangements allowing for the interaction between the NBD and MD result in the closed conformation which is able to undergo hydrolysis. Following hydrolysis, ADP and Pi
are released and Hsp90 returns to the open apo-state. This model does not exclude the possibility of a distinct ADP-bound conformation [12
] following hydrolysis as it does not contribute to the rate-limiting step of the hydrolysis reaction, which has been shown for hHsp90 by kinetic and single-turnover experiments to occur after nucleotide binding but before hydrolysis [9
As was mentioned previously, the binding of co-chaperones to eukaryotic Hsp90 can result in specific conformations that are necessary for driving the chaperone cycle through completion [1
]. Their role as regulators of the cycle has been enhanced in light of single molecule FRET experiments which have shown that in the absence of co-chaperones or substrate molecules, ATP hydrolysis is not tightly coupled to the conformational cycle [5
]. It appears that conformational states of Hsp90 can quickly and reversibly change without committing to hydrolysis and that the co-chaperones function to stabilize a conformation required for progression through the ATPase cycle. Chaperones modulate Hsp90 function by altering ATP turnover or by facilitating client loading and activation. The co-chaperones Cdc37 and HOP are both involved in the recruitment of client proteins and are able to arrest the ATPase cycle of Hsp90 in order to facilitate client protein loading. Cdc37 slows down the ATPase cycle by binding to sites on the lid segment of the N-terminal domain in the open conformation, fixing the ATP lid in an open conformation and preventing interaction of the N-domains [20
]. HOP functions by coupling the Hsp70 and Hsp90 chaperones and facilitates client protein transfer between the two. HOP prevents N-terminal dimerization by binding to the open conformation of Hsp90 [21
]. p23 slows down the ATPase cycle by binding to and stabilizing the ATP-bound closed conformation which is essential for activation of client proteins [13
]. To date, only one activator of the ATPase activity of Hsp90, Aha1 (activator of Hsp90 ATPase1), is known which has been shown to stimulate activity by a factor of 100 or more [9
]. Aha1 binds to the open conformation of Hsp90 at both the N terminal and MDs, inducing a conformational change resulting in N-terminal dimerization and stabilization of the ATPase-competent conformation [22
]. Interestingly, the binding of only one Aha1 molecule is necessary to fully stimulate ATPase activity and results in an asymmetric complex [22
]. Aha1 appears to enhance ATPase activity by reducing the energy barrier accompanying structural rearrangements that occur during the transition between the open and closed states, which have been shown to be rate limiting [4
While it is still unclear precisely how Hsp90 induces client protein conformational changes, it is likely that it is directly linked to the domain movements and conformational changes that occur to Hsp90 as it goes from the ‘closed’ to ‘open’ conformational states. The first structural insight into client protein interaction with Hsp90 was provided by Vaughan et al.
] who used single-particle EM to determine the structure of Hsp90–Cdc37–CDK4 complex. CDK4 is a protein kinase that is dependent on Hsp90 for activation and on Cdc37 for recruitment [24
]. This structure shows that client interactions occur to both the MD and NBD of one Hsp90 subunit while Cdc37 binds to the NBD of the other subunit. While not proven, the fact that this complex contains Cdc37 may suggest that binding of client to Hsp90 occurs before the catalytically competent ATP-bound conformation, which requires that Cdc37 disengage from the complex [23