Molecular Cell

Article

Species-Dependent Ensemblesof Conserved Conformational StatesDefine the Hsp90 Chaperone ATPase CycleDaniel R. Southworth1 and David A. Agard1,*1Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California, San Francisco,San Francisco, CA 94158, USA*Correspondence: agard@msg.ucsf.eduDOI 10.1016/j.molcel.2008.10.024

SUMMARY

The molecular chaperone heat shock protein 90(Hsp90) is required for the folding and activation ofnumerous essential signaling proteins. Hsp90 is gen-erally thought to transition between an open (apo)and a closed (ATP) conformation in response tonucleotide. Here, 3D single-particle reconstructionsof Escherichia coli and yeast Hsp90 homologs estab-lish the existence of two distinct nucleotide-stabi-lized conformations (ATP, ADP) in addition to anapo extended state, supporting previous structuralwork. However, single-particle matching methodsreveal that, rather than being irreversibly determinedby nucleotide, a species-dependent dynamic confor-mational equilibrium exists between states. Usingcrosslinking methods, we trap transient nucleotide-specific states of yeast and human Hsp90 and estab-lish that the apo, ATP, and ADP states are universal.These data support a conserved three-state chaper-one cycle where the conformational equilibriumvaries between species, implicating evolutionarytuning to meet the particular client protein and meta-bolic environment of an organism.

INTRODUCTION

The diverse class of proteins known as molecular chaperonesplays essential roles in maintaining the population of correctlyfolded proteins in the cell. This generally occurs by recognitionof exposed hydrophobic regions on the unfolded substratesand repeated rounds of ATP-dependent binding and release(Hartl and Hayer-Hartl, 2002; Young et al., 2004). Well-describedchaperones include the heat shock proteins 70 (Hsp70) (Zhuet al., 1996) and 60 (GroEL) (Martin et al., 1991), which act earlyin the folding process to stabilize nascent polypeptide chainsand facilitate the folding of relatively unstructured intermediates(Bukau and Horwich, 1998). By contrast, the abundant heatshock protein 90 (Hsp90) family preferentially interacts with itssubstrate (client) proteins late in the folding pathway, facilitatingessential protein-protein and protein-ligand interactions (Nathanet al., 1997; Picard, 2002; Zhao et al., 2005). Despite character-

ization of numerous client proteins that require Hsp90-mediatedactivation, much of the conformational rearrangements, proteininteraction sites and nucleotide dependence of the chaperoneprocess remain unclear.Hsp90 is highly conserved from Escherichia coli (HtpG) to

yeast (Hsc82 and Hsp82 isoforms) to humans (Hsp90a andHsp90b isoforms). Although client proteins have not been defin-itively identified for HtpG, in higher organisms Hsp90 activity isessential for the activation of many proteins including steroidhormone nuclear receptors (Picard et al., 1990; Pratt et al.,2004), protein kinases (Richter and Buchner, 2001; Sato et al.,2000), nitric oxide synthase (Garcia-Cardena et al., 1998), andtelomerase (Holt et al., 1999). For eukaryotes, both client-specific and general cochaperones are required during thechaperone process (Felts and Toft, 2003; Johnson et al., 1998).Hsp90-specific inhibitors, including geldanamycin and its deriva-tives, promote the inactivation and degradation of numerous cli-ent oncoproteins and cell cycle kinases (Neckers, 2002; Roeet al., 1999).Hsp90 contains three distinct domains: a C-terminal, high-

affinity dimerization domain (CTD), a middle domain (MD) impor-tant for substrate maturation, and an N-terminal ATPase domain(NTD). Crystal structures of the ATPase domain identified it asa member of the GHKL family that includes DNA gyrase and top-oisomerase II (Dutta and Inouye, 2000; Prodromou et al., 1997).From comparisons of these family members and biochemicalstudies, it has been proposed that the energy from nucleotidebinding triggers a conformational switch where the NTDs dimer-ize to form a closed state that is required both for hydrolysis andclient protein activation (Ban et al., 1999; Prodromou et al.,2000). Although distinct client binding sites have yet to be iden-tified, regions of significant exposed hydrophobic surface areawithin each domain indicate potential interaction sites (Shiauet al., 2006; Harris et al., 2004;Meyer et al., 2003). Notably, a neg-ative-stain electron microscopy (EM) reconstruction of a yeastHsp90:Cdc37:Cdk4 complex shows theCdk4 kinase client inter-acting with a single monomer near the NTD, suggesting a moreasymmetric mode of client interaction (Vaughan et al., 2006).Crystal structures of intact Hsp90 homologs from yeast (Ali

et al., 2006), E. coli (Shiau et al., 2006) andmammalian endoplas-mic reticulum (Dollins et al., 2007) reveal dramatically differentconformations that result from large rigid-body movements ofthe domains. The structure of apo HtpG identified an open con-formation with a large CTD-MD angle and an NTD arrangement

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that results in an 80 A separation of N-terminal residues. TheGrp94 structures have essentially the same NTD conformation,but a different open, CTD-MD angle. The crystal structure ofyeast Hsp82 bound to AMPPNP (nonydrolyzable ATP analog)and in a complex with the p23 cochaperone identified a closed,‘‘ATP’’ conformation, supporting models from previous bio-chemical work (Prodromou et al., 2000). This closed conforma-tion has a reduced CTD-MD angle compared with either apoHtpG or Grp94 and a 90! rotation of the NTD resulting in dimer-ization of the NTDs. For HtpG, ADP binding also resulted in a sig-nificant conformational change, yielding a unique tetramer in thecrystallographic asymmetric unit. The packing arrangementalong with 2D EM data led to a model for a compact, ‘‘ADP’’ di-mer that retained the large buried surface area (3700 A2 permonomer) observed in the crystal form.

Recent solution small-angle X-ray scattering (SAXS) experi-ments with HtpG have identified additional complexity by mod-eling an apo conformation that is more extended than the crystalform and a mixture of open and closed states in the presence ofAMPPNP, but no changes were seen with ADP compared withapo (Krukenberg et al., 2008). Cryo-EM and SAXS studies of hu-man Hsp90 showed two different apo conformations (Bron et al.,2008), whereas no significant conformational changes havebeen observed in the presence of nucleotide (Zhang et al.,2004). Thus, whereas previous structural work illustrates sub-stantial domain rearrangements of Hsp90, critical questionsremain about the relevant conformational states, including thenucleotide dependence, the extent to which they are populatedduring the chaperone cycle and the degree of conservationacross species.

Although challenging because of the small size of Hsp90,single-particle EM is particularly well suited to address thesequestions because it provides direct visualization of structuralstates unconstrained by crystal lattice contacts better reflectingthe solution state of Hsp90. In the following work we investigatethe nucleotide-dependent conformational states of E. coli, yeast,and human Hsp90s. We provide 3D reconstructions ofHtpG:AMPPNP and HtpG:ADP that support a model for the hy-drolysis cycle involving three distinct states: an extended apostate, a closed ATP state, and a compact ADP state. Our recon-struction of yeast Hsp90 (Hsc82):AMPPNP establishes that nu-cleotide binding alone drives NTD dimerization. Detailed com-parison with the crystal structure suggests an altered NTD-MDconformation in the absence of p23 that reflects an active stateon-pathway to hydrolysis. Through crosslinking, we were able totrap transiently populated states and identify the closed ATPconformation in human Hsp90 and the compact ADP conforma-tion in both human and yeast Hsp90s.

Finally, our work reveals that the Hsp90 nucleotide cycle oper-ates quite differently than related GHKL family members wherenucleotide binding triggers a discrete conformational change.Analysis of single-particle images reveals that the Hsp90 confor-mational states are not deterministic; rather, the different statescoexist in a dynamic equilibrium. Nucleotide binding is found toprovide only modest stabilizing energy, biasing the equilibriumtoward, but not dictating, the conformational state. This worksupports amodel involving a universal three-state Hsp90 confor-mational cycle, where the equilibrium between states is species

dependent and thus evolutionarily optimized to meet the de-mands and influences of different client proteins and cochaper-ones found in each organism.

RESULTS

Species-Dependent Occupancy of Open, Closed,and Compact Conformations of Hsp90Previous structural studies identified dramatically different con-formational states of Hsp90 but raised critical questions aboutthe nucleotide dependence and the conservation of thesesstates between species. Therefore we sought to determinehow nucleotide binding alone affects the conformational stateof E. coli, yeast, and human Hsp90s using negative-stain EM.Recombinantly expressed and purified HtpG (E. coli), Hsc82(yeast), and Hsp90a (human) were incubated in the presenceor absence of saturating concentrations of AMPPNP and ADP.From the example particles (Figure 1) and micrographs (see Fig-ures S1, S2, and S3 available online) it is clear that HtpG adoptsthree distinct nucleotide dependent conformations and Hsc82adopts two conformations, whereas for Hsp90a no significantchanges are observed. In the absence of nucleotide HtpG,Hsc82 and Hsp90a adopt a heterogeneous ensemble of openand extended conformations with an overall shape that is consis-tent with previous studies (Krukenberg et al., 2008; Bron et al.,2008). Interestingly, in micrographs of apo HtpG, other low-pop-ulation conformations that appear more compact in shape canbe identified. These are reminiscent of the proposed nucleo-tide-bound conformations (see below).In the presence of AMPPNP, both HtpG and Hsc82 adopt

a significantly different, closed conformation that appears tomatch the yeast Hsp82:AMPPNP:p23 crystal structure (Aliet al., 2006). From themicrographs (Figures S1 andS2), amixtureof open and closed particles is observed for HtpG but for Hsc82a significantly greater fraction appear closed. Conversely, whenHsp90a is incubated with AMPPNP no conformational changesare observed (Figures 1 and S3). Incubations with ATP andATPgS also resulted in no apparent changes in Hsp90a confor-mation (data not shown). Experiments performed with theHsp90b isoform gave identical results to Hsp90a (data notshown). Finally, in the presence of saturating amounts of ADP,HtpG adopts a third, highly compact conformation that differssignificantly from apo HtgG and HtpG:AMPPNP. TheseHtpG:ADP particles look more globular and similar to the com-pact dimer conformation modeled from the HtpG:ADP crystalstructure. By contrast, no significant conformational changesare observed when either Hsc82 or Hsp90a is incubated withADP.These results establish that the extended, apo conformation is

conserved, that AMPPNP binding alone stabilizes a closed con-formation in HtpG and Hsc82, and that ADP binding results in analternate, highly compact conformation in HtpG. What is unclearfrom these 2D views is how similar the closed, AMPPNP-boundconformations are to the Hsp82:AMPPNP:p23 crystal structureandwhether theHtpG:ADP particles closelymatch the proposedADPmodel.Finally, theobservation thatnoconformationalchangeswere seen with Hsp90a:AMPPNP, Hsp90a:ADP, and Hsc82:ADPindicates that there are fundamental species-dependent

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differences and suggests that either these states are not uni-versal or that they are only transiently sampled.

Three-Dimensional EM Reconstructionsof HtpG:AMPPNP and HtpG:ADP Identify DistinctClosed and Compact ConformationsOur 2D images of HtpG:AMPPNP and HtpG:ADP are strikinglydifferent than those of apo HtpG, revealing significant nucleo-tide-dependent conformational changes. However, detailed 3Danalysiswas required toproperlydistinguish theseconformationsand make comparisons to the observed Hsp82:AMPPNP:p23and modeled HtpG:ADP structures. Therefore, we performed3Dsingle-particle reconstructions.An initial setofHtpG:AMPPNPparticles were collected for reference-free class averaging (Fig-ures 2A and S4A). Given the two-fold symmetry of the crystalstructures of Hsp90, the same symmetry was imposed for theinitial model (Figure S4B) and during the refinement. The resolu-tion of the final model was estimated to be 22 A using the Fouriershell correlation method (FSC = 0.5 for all reconstructions)(Figure S6A).In the HtpG:AMPPNP reconstruction, two large globular do-

mains, predicted to be the NTDs, appear dimerized along the in-side surface and separate into extended middle domains, thenreconnect in a smaller globular domain (Figures 2B and 2C). Asurprisingly large cavity (roughly 55 by 15 A) that was not appar-ent from the reference-free averages separates the two middledomains. The yeast Hsp82:AMPPNP:p23 crystal structure(Figure 2D) and a model based on the HtpG sequence (datanot shown) both fit well into the 3D reconstruction. The fit ofthe structures confirms the proposed architecture for theHtpG:AMPPNP reconstruction and identifies a closed conforma-

tion indistinguishable from the yeast Hsp82:AMPPNP:p23 struc-ture. Regions that are outside the density (when contoured to themolecular weight) include flexible loops at the CTD-MD junctionand the flexible amphipathic helices in the CTD. Importantly, this3D reconstruction of HtpG:AMPPNP establishes that the closedATP conformation is conserved in E. coli and that nucleotidebinding alone is sufficient to drive a large-scale rearrangementresulting in NTD dimerization.Initial 2D views of HtpG:ADP reveal a distinct compact confor-

mation supporting our previous work. Reference-free class aver-ages confirmed the globular shape (typical dimensions of 60 by95 A) (Figure 3A). From the averages an initial model was ob-tained using the cross common lines method with two-fold sym-metry imposed (Figures S4C and S4D). The 2D projections of thefinal model and the corresponding class averages match welland present an overall compact trapezoid shape (Figure 3B).The resolution of the final model was estimated to be 23 A(Figure S6B).Our reconstruction illustrates that HtpG:ADP has adopted

a conformation that is much more compact than HtpG:AMPPNP

Figure 1. Three Nucleotide-Stabilized Conformational States ofHsp90 Vary between SpeciesTypical 2D negative stain single-particle views of E. coli HtpG, yeast Hsc82,

and human Hsp90a are shown following incubations in the absence (apo) or

presence of AMPPNP and ADP. Box sizes equal 330 A.

Figure 2. Three-Dimensional Reconstruction of HtpG:AMPPNPReference-free class averages are shown (A), with the number of single parti-

cles used to generate the averages listed on each image. A set of projections

and class averages for the final model (B) are shown. Box sizes equal 250 A. In

(C) the final HtpG:AMPPNP 3Dmap is shown, and in (D) the yeast Hsp90 struc-

ture (Ali et al., 2006) was fit into the EMmap. The monomers of the homodimer

are colored green and brown.

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(Figure 3C). Higher-density regions appear to be the NTDs thathave rotated down, making alternate intermonomer contacts.The HtpG:ADP compact model predicted from our tetramerHtpg:ADP crystal structure (Shiau et al., 2006) fits with significantaccuracy into the EM volume (Figure 3D). The overall shape ofthe EM map and the docked model indicates that these struc-tures are essentially identical, establishing the existence ofa compact ADP-dependent conformation that is dramatically dif-ferent than the ATP-bound state for Hsp90.

Three-Dimensional EM Reconstruction of YeastHsc82:AMPPNP Establishes Nucleotide-DrivenNTD DimerizationThe 2D images of Hsc82:AMPPNP established that nucleotide-binding alone promotes a closed conformation in yeast Hsp90(Figure 1). To ascertain the role of p23 in determining the closedconformation, it was necessary to obtain a complete single-particle 3D reconstruction and compare it with the Hsp82:AMPPNP:p23 crystal structure. Reference-free class averageswere generated from an initial data set and clearly resemblethe HtpG:AMPPNP averages (compare Figure 4A andFigure 2A). Therefore, the HtpG:AMPPNP initial model (FiguresS4A and S4B) was used for the Hsc82:AMPPNP reconstruction.A set of projections and corresponding class averages of the fi-nal round of refinement are shown (Figure S5A) and the resolu-

tion was estimated to be 24 A (Figure S6C). The reconstructionis quite similar to the Hsp82:AMPPNP:p23 structure, clearlydemonstrating the dimerized, globular NTDs, an extended MD,and a CTD that is better resolved than for the HtpG reconstruc-tion (Figure 4B). As expected, the Hsp82:AMPPNP:p23 crystalstructure fits extremely well in the density. This fit is an improve-ment over that found for the HtpG:AMPPNP reconstruction, witha larger fraction of atoms fitting within the EM map (0.72 com-pared with 0.66 for HtpG:AMPPNP). Interestingly, the span ofdensity between the NTDs is reduced compared with the crystalstructure, indicated by portions of the NTD and middle domainsthat are outside the EM volume at the appropriate contour.To better address the potential for an altered NTD-middle do-

main arrangement, we calculated a differencemap of the alignedHsp82:AMPPNP crystal structure and our 3D reconstruction(Figure 4C). This facilitates the identification of nonoverlappingregions, indicating a different arrangement between the crystalstructure and our reconstruction. In the difference map (experi-mental " calculated), positive density (blue, Figure 4C, upper

Figure 3. Three-Dimensional Reconstruction of HtpG:ADPReference-free class averages (A) and example projections with correspond-

ing averages for the final model (B) are shown. The box sizes are equal to

210 A. The final HtpG:ADP 3Dmap (C) and the compact HtpG structure model

(Shiau et al., 2006) fit into the EM map (D) are shown, identifying the compact

conformation.

Figure 4. Three-Dimensional Reconstruction of Hsc82:AMPPNPReference-free class averages are shown in (A). In (B) the final Hsc82:AMPPNP

reconstruction is shown along with the Hsp82:AMPPNP crystal structure

docked into the map. In (C) a difference map (experimental " calculated) is

shown aligned to the yeast Hsc82:AMPPNP reconstruction. In orange is den-

sity present in the crystal structure but missing in the EM volume, and blue is

additional EM map density. For comparison, the yeast Hsp82:AMPPNP:p23

complex is shown (middle panel). Two p23 molecules (raspberry) flank the

NTD dimerization interface. An improved, rigid-body docking of the domains

into the EM map (lower panel) highlights an alternate closed conformation

with a 12! rotation of the NTD.

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panel) is present in several small regions near the CTD. Interest-ingly, significant negative density (orange), representing regionsof the crystal structure that are not present in the reconstruction,spans the area between the monomers at the base of the NTDs.From the structure of the Hsp82:AMPPNP:p23 complex it isclear that p23 interacts directly in this region (Figure 4C, middlepanel), likely providing additional stabilizing interactions alongthe NTD dimerization interface. As a control we performed a re-construction with the total data set using the crystal structurevolume as the initial model (low-pass filtered to 30 A). After eightrounds of refinement the data converged to a model identical towhen the common-lines generated model was used, eliminatingmodel bias in our analysis and further substantiating the differ-ences between our final model and crystal structure (data notshown).We performed rigid body fitting of flexible domains to deter-

mine if an altered NTD-MD arrangement could better describethe Hsc82:AMPPNP reconstruction. This method significantlyimproved the fit; the fraction of atoms within the EM density in-creased from 0.72 to 0.74 and the cross-correlation value im-proved from 0.60 to 0.63 (Figure 4C, lower panel). The primarystructural change was a rotation of 12!about the NTD-MD inter-face, extending the NTDs slightly up and outward with an overalluntwisting of the dimer. Some MD density still remains outsidethe map, but fits appropriately at a lower contour threshold (Fig-ure S5B). Thus, our improved fit presents a plausible alternate ar-rangement of the closed yeast Hsc82:AMPPNP dimer, onewhere p23 is not providing additional stabilizing interactions.

Electron Microscopy Analysis of the Equilibriumof Hsp90 Conformational StatesDuring ATP binding, Hsp90 is generally thought to convert froman open to closed conformation through a two-state cycle. How-ever, our recent SAXS studies demonstrated more nuanced be-havior in solution: whereas apo HtpG primarily exists in an openand extended state, in the presence of AMPPNP only a portion ofthe population shifts to a closed conformation (Krukenberg et al.,2008). By providing direct views of individual single particles, EMimages allow us to better determine relative populations of theconformations in different nucleotide states. This in turn yields in-sight into hownucleotide binding energy is utilized to drive a pop-ulation of molecules to an alternate conformation.As discussed above, HtpG primarily adopts an open, ex-

tended arrangement in the absence of nucleotide (Figure 1).However, additional closed conformations were also observedat a low frequency. Further analysis reveals that these closedparticles match quite well with the closed ATP and compactADP forms of HtpG (Figures 5A and 5B). This is striking becauseit indicates that in the absence of the binding energy fromAMPPNP or ADP, HtpG is able to sample these alternate confor-mations. Thus, these data suggest that in the absence of nucle-otide, HtpG adopts an ensemble of states that reflect thecomplete nucleotide cycle.Ideally we would like to quantitatively explore species-depen-

dent differences in the Hsp90 conformational equilibrium. How-ever, because there were no visually discernable differencesbetween Hsc82:ADP, Hsp90a:AMPPNP, and Hsp90a:ADP andtheir respective apo states, we chose to focus on HtpG and

Hsc82 and determine the conformational equilibrium of their ex-tended apo and closed ATP states. A multireference-based ap-proach was used in which single particles of HtpG and Hsc82were matched to either the open or closed conformation. The2D projections of the apo HtpG and closed Hsp82:AMPPNPmodels were generated and first aligned to each other to deter-mine, based on a correlation value, similar and distinct views.Single-particle data sets of apo and AMPPNP-bound HtpGand Hsc82 were then collected and scored against the totalset of references. To avoid ambiguous views, only particle im-ages that showed a distinct preference for either the open orthe closed model were used in calculating the fraction of theopen and closed states.Overall, the data reveal two main points (Figure 5C): (1) both

HtpG andHsc82 exhibit an incomplete shift to the closed confor-mation in the presence of nucleotide; and (2) the conformationalequilibrium is significantly different between the two Hsp90s,with a more significant population shift from the open to closedstate for Hsc82. For HtpG, in the absence of nucleotide, 68%

Figure 5. Single-Particle Analysis Identifies an Equilibrium betweenthe Different Conformational States(A) A typical micrograph showing multiple conformations of apo HtpG.

Extended (blue), closed ATP-like (green), and compact ADP-like (orange) par-

ticles are boxed and enlarged in (B) along with comparable views of the corre-

sponding structures. Scale bar equals 500 A, and the box sizes are 330 A.

(C) A set of single particles of HtpG or Hsc82 in the absence or presence of

AMPPNP were collected and matched to a set of unique 2D projections of ex-

tended apo and closed ATP structures to determine the fraction of extended

(dark gray) and closed (light gray) conformations. Error bars shown represent

the standard deviation of three independent experiments.

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of the particles match the extended conformation, whereas 32%match the closed ATP conformation. This essentially reverses toa 33% open and 67% closed when AMPPNP is present. Thedata for Hsc82 show that vast majority of the particles (82%and 84%, respectively) match either the open conformation inthe absence of nucleotide or the closed conformation whenAMPPNP is present, indicating a more substantial equilibriumshift to the closed state.

Crosslinking Establishes that the ATP and ADPConformational States Are Universal to Hsp90Despite significant sequence and structural conservation, theclosed conformation has not been observed with humanHsp90. In fact, previous studies have suggested that for humanHsp90 the NTDs act independently, possibly not dimerizing dur-ing the nucleotide cycle (McLaughlin et al., 2004). Furthermore,our results here failed to reveal any significant conformationalchanges in human Hsp90a in the presence of saturatingamounts of AMPPNP (Figure 1). However, recent kinetic studiessupport a conserved ATPase cycle and show evidence for NTDdimerization in human Hsp90 (Frey et al., 2007; Richter et al.,2008). Given these conflicting studies and our observation thatHtpG and Hsc82 have dramatically different conformationalequilibria, we hypothesized that although a closed conformationcan exist for human Hsp90:ATP, the conformational equilibriummust be strongly shifted toward the open state. Thus, in the pres-ence of nucleotide only a small fraction of human Hsp90 wouldexist in the closed conformation.

In order to trap rarely populated conformational states, wecrosslinked Hsp90a by treating samples with a small amountof glutaraldehyde (0.005%) in the presence or absence of nucle-otide before visualization. Remarkably, when Hsp90a is cross-linked after incubation with AMPPNP, numerous closed particlespredominate and the class averages appear to be identical to theHtpG and Hsc82 closed conformation (Figures 6A and S7C). In-cubation with glutaraldehyde in the absence of nucleotide doesnot produce significant numbers of closed particles, indicatingthat this closed state is nucleotide dependent (Figures S7Aand S7B). Interestingly, the apo crosslinked particles appear cir-cular in shape, with a small NM angle and an open MC angle, re-sulting in a conformation that is distinct from the ATP and ADP

states. This conformation is comparable to the ‘‘semi-open’’ ar-rangement of mammalian apo Hsp90 identified in recent cryo-EM studies (Bron et al., 2008). A low population (<5%) of closedHsp90a ATP-like conformations was observed in the apo cross-linked data, suggesting that the closed state is also sampled ata very low level in the absence of nucleotide (data not shown).Importantly, these data reveal that although human Hsp90a ex-ists predominantly in the open conformation in the presence ofAMPPNP, the closed ATP state is clearly sampled in a nucleo-tide-dependent manner. This provides structural evidence thathumanHsp90a forms the closed state and supports a conservedconformational change during the nucleotide cycle.Given the success of this method in revealing the closed con-

formation in Hsp90a, crosslinking was attempted in the pres-ence of ADP. Hsp90a was incubated with saturating amountsof ADP along with low concentrations of glutaraldehyde as be-fore, followed by negative staining. From the micrographs andclass averages Hsp90a:ADP appears highly compact and iden-tical to projections of the HtpG:ADP reconstruction (Figures 6Band S7D). Next, we crosslinked yeast Hsc82 in the presence ofADP and, strikingly, Hsc82 also appears highly compact andidentical to HtpG:ADP (Figures 6C and S8B). As a control,Hsc82 alone was also incubated in the presence of glutaralde-hyde and no significant differences were observed comparedwith the absence of crosslinker (Figure S8A). These results iden-tify that, as with HtpG, both human Hsp90a and yeast Hsc82transiently form a compact conformation in an ADP-dependentmanner. The nucleotide dependence clearly establishes thatthe compact ADP conformation is a distinct and well-conservedconformational state that occurs during the hydrolysis cycle ofHsp90 and, as with the ATP state, the conformational equilibriumis species dependent.

DISCUSSION

Recent full-length crystal structures of different Hsp90 homologshave revealed identical domain architecture but dramatically dif-ferent conformational states. Coupled to these conformations,the nucleotide cycle of Hsp90 has been thought to be analogousto other related GHKL family members, where ATP bindingdrives a discreet conformational switch from an open to closed

Figure 6. Crosslinking Reveals the ClosedATP and Compact ADP Conformations inHuman and Yeast Hsp90(A) Hsp90a was incubated with AMPPNP and

0.005% glutaraldehyde crosslinker before nega-

tive staining. Aligned single images, reference-

free class averages, and aligned 2D projections

of the Hsc82:AMPPNP reconstruction are shown

(right to left). Box sizes are 250 A.

Hsp90a (B) and yeast Hsc82 (C) incubated with

ADP and crosslinker are shown as in (A) and com-

pared with the HtpG:ADP reconstruction.

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state. The differences in the structures, the speculative nature ofthe HtpG ADP state, and the failure to observe some conforma-tional states have raised significant questions including the con-servation between species and the degree to which nucleotidestabilizes the different states. Here we have investigated theapo and nucleotide-bound conformations of the E. coli, yeast,and human Hsp90 homologs using single-particle EM methods.Our results reveal a unique conformational cycle for Hsp90, in-volving at least three dramatically different states that exist inequilibrium. Unexpectedly, we found that nucleotide bindingonly provides modest stabilization energy and that the occu-pancy of the different states is highly species dependent. Our re-sults with human Hsp90 illustrate the most striking difference:nucleotide binding (ATP or ADP) does not substantially shiftthe conformational equilibrium away from the apo state, unlikeE. coli and yeast Hsp90. However, identified by our crosslinkingresults, the ATP-closed and ADP-compact nucleotide-stabilizedstates indeed exist for human Hsp90, establishing a universalthree-state conformational cycle.

The Closed, NTD Dimerized Conformation of Hsp90Is Conserved from E. coli to Humans and Stabilizedby ATP BindingOur 3D reconstructions of HtpG:AMPPNP and Hsc82:AMPPNPidentify that AMPPNP binding alone stabilizes a closed confor-mation that closely matches the yeast Hsp82:AMPPNP:p23complex. Furthermore, crosslinking analysis of Hsp90a revealsthat this conformation is conserved in human Hsp90s as well,validating previous experiments identifying that human Hsp90can compliment yeast Hsp90 function in vivo (Picard et al.,1990). From our reconstructions and the accurate fit of the crys-tal structure, it is clear that ATP binding induces a large-scale ro-tation of the NTDs from the orientation observed in either theapo-HtgG or Grp94 structures. This closed conformation hasbeen identified as catalytically significant and involves a directinteraction between a middle domain arginine residue and theg-phosphate of ATP (Ali et al., 2006; Meyer et al., 2003). The re-constructions further support the importance of the closed con-formation in the hydrolysis cycle by identifying that the energyfrom nucleotide binding alone is sufficient to drive the large con-formational rearrangements in Hsp90, establishing an essentialrole for nucleotide in the chaperone mechanism.In the yeast Hsp82:AMPPNP:p23 crystal structure the closed

conformation is clearly stabilized by two p23 cochaperones in-teracting adjacent to the NTD dimerization interface. This inter-action is known to significantly slow the hydrolysis rate and sta-bilize client-bound Hsp90 complexes (McLaughlin et al., 2006).In our 3D reconstruction of Hsc82:AMPPNP we observe differ-ences compared to the crystal structure of the p23-bound com-plex that reflect a different arrangement, where the NTDs haverotated up and away slightly. This structural evidence for an al-ternate, closed conformation suggests a model where the p23-bound state is off-pathway for ATP hydrolysis. In support ofthis idea, biochemical work from our lab has identified a T22Fmutation in Hsc82 that results in a significant increase in thehydrolysis rate but is sterically incompatible with theHsp82:AMPPNP:p23 structure (Cunningham et al., 2008). Inthe crystal structure this mutation is located in a helix that is

sandwiched between p23 and the opposing NTD and is pre-dicted to destabilize NTD dimerization. Therefore, a conforma-tional change where the NTDs rotate away slightly followingp23 release may be required for maximum hydrolysis activity.This potential mechanismwould be addressed from high-resolu-tion structures of Hsc82:AMPPNP alone or possibly with Aha1,a cochaperone that interacts nearby and stimulates ATP hydro-lysis (Meyer et al., 2004).

The Compact ADP Conformation Represents a ThirdState in the Hydrolysis CycleOur 3D reconstruction of HtpG:ADP clearly establishes theexistence of a more compact conformation that likely results fol-lowing ATP hydrolysis. Remarkably our newly determined 3Dvolume quantitatively matches the compact dimer model pro-posed based on domain-swapping of the observed crystallo-graphic tetramer. From comparisons of this crystal structuremodel and other Hsp90 structures, it is clear that the ADP con-formation involves a significant rotation of the NTD down towardthe MD whereas the MD-CTD angle closely matches the closedATP conformation. In support of this arrangement, previous bio-chemical studies on Grp94 identified interdomain crosslinks thatare only consistent with the domain geometry of the ADP-com-pact state (Chu et al., 2006).Because the vast majority of the HtpG:ADP molecules are in

this conformation in our EM micrographs, this appears to bean authentic stable state in the E. coli Hsp90 ATPase cycle. Bycontrast, Hsc82 and Hsp90a do not form the compact state toan appreciable degree under similar conditions. Furthermore,this conformation has not been observed by solution SAXS anal-ysis from our lab (HtpG) and others (human) (Zhang et al., 2004),leading to questions about its existence. Given our EM data, weargue that experimental differences including the 1000-foldhigher protein concentrations necessary in SAXS experimentsand possible transient higher-order interactions (Chadli et al.,1999) might deter formation of the dimeric ADP state. More im-portantly, just as crosslinking established the existence of atransient closed Hsp90a:AMPPNP state, it also confirms thatthe dimeric ADP conformation is indeed populated in an ADP-dependent manner for both human and yeast Hsp90s. Thesedata establish that the ADP state is in fact conserved among dif-ferent organisms, supporting a functional role in the chaperonecycle.

Model for the Hsp90 Hydrolysis CycleFrom the work presented here we propose a model for theHsp90 chaperone cycle that involves three specific conforma-tional states that are in a dynamic and species-dependent equi-librium (Figure 7). ATP binding and hydrolysis provide modestamounts of stabilization energy and help move Hsp90 throughthe cycle. In the absence of nucleotide, Hsp90 predominantly ex-ists in an open conformation (1) that is significantly more ex-tended than the apo-HtgG crystal structure and variable in theopen angle, supporting recent cryo-EM data (Bron et al.,2008). Importantly, apo Hsp90 also samples the closed andcompact states (Figure 5; Figure 7, 1b and 3) indicating the con-formational equilibrium exists in the absence of nucleotide. ATPbinding biases this equilibrium to the closed state (2); however,

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Molecular Cell 32, 631–640, December 5, 2008 ª2008 Elsevier Inc. 637

the shift is drastically different among the three species ofHsp90s studied here. In E. coli the conversion to the closed stateis modest, in yeast there is a dramatic shift, and in human Hsp90very little change occurs. These results highlight potential differ-ences in the utilization of ATP binding energy in stabilizing theclosed conformation. Binding of p23 further stabilizes a closedHsp90 but restricts NTD-MD rotations necessary for hydrolysis(Figure 7, 2b). After hydrolysis, Hsp90 transiently adopts thecompact conformation (3) and then cycles back to the open, ex-tended state.

Finally, the different conformational equilibria we have identi-fied point to optimization of the kinetic cycle within different or-ganisms while the overall chaperone mechanism remains con-served. These observed equilibrium differences parallel theknown differences in hydrolysis rates as well, with the fastestturnover observed for yeast Hsp90 (1.3 3 10"2 s"1; Prodromouet al., 2000), a moderate rate for HtpG (#5 3 10"3 s"1; C.N.Cunningham and D.A.A., unpublished data) and a very lowrate for human Hsp90 (1.5 3 10"3 s"1; McLaughlin et al.,2002), suggesting that NTD rearrangement and dimerization israte limiting for the overall hydrolysis cycle. Interestingly, ourdata indicate that the energetics of NTD dimerization are quitedifferent between organisms. For HtpG, dimerization appearsto be relatively isoenergetic with nucleotide binding producingonly a slight increase in the fraction of closed complexes andthus a very small DG for the closed conformation. For yeast,the equilibrium data suggest that the DG is much larger, with nu-cleotide binding resulting in a significant shift to the closed state,but for human Hsp90 the conformational change remains unfa-vorable. In vitro experiments have identified a higher humanHsp90 ATPase rate in the presence of a known client protein(McLaughlin et al., 2002). Therefore, one possible reason forthese differences is that for human Hsp90, productive clientprotein interactions along with nucleotide binding stabilize theclosed state. Additional structural and biochemical experimentswill be critical in determining whether the interactions made byclient proteins and cochaperones contribute to the conforma-tional equilibrium and how these dramatic conformationalchanges promote stabilization and activation of specificsubstrates.

EXPERIMENTAL PROCEDURES

Protein Expression and PurificationFull-length HtpG (E. coli), cloned into a pET29b vector (Novagen) and under the

control of an IPTG-inducible T7 promoter, was expressed in BL21 cells (Invitro-

gen). Full-length HtpG and Hsc82 protein were purified as described previously

(Cunningham et al., 2008; Krukenberg et al., 2008). Human Hsp90a DNA, pro-

vided by the David Toft lab, was cloned into the pET151 vector containing a 63

His-tag andTEVcleavagesite (Invitrogen).Hsp90aproteinwaspurifiedby ionex-

change (DE52 resin, Whatman), Ni-NTA affinity, and size exclusion (Sephacryl

S-200 column, GE Healthcare), followed by TEV cleavage of the His-tag. We as-

sessed the quality of each purification by measuring the ATP hydrolysis rates

(Cunningham et al., 2008) and comparing them with known or published rates.

Electron MicroscopyHtpG, Hsc82 and Hsp90a proteins were negatively stained with uranyl formate

(pH 5.5–6.0) on thin carbon-layered (40–50 A thick) 400 mesh copper grids

(Pelco) essentially as described previously (Ohi et al., 2004). Initial experiments

were performed with other stains including ammonium molybdate, methyl-

amine vanadate, and uranyl acetate. The particles appeared similar; however,

the low contrast and granularity prevented further analysis. Before staining,

protein samples were typically incubated for 20 min at 150 nM with or without

5 mM AMPPNP or 2mM ADP (Sigma-Aldrich) at 37! for HtpG and Hsp90a and

30! for Hsc82 in an incubation buffer containing 20 mM Tris (pH 8.0), 20 mM

KCl, 5 mM MgCl2 and 1 mM DTT. KCl and MgCl2 concentrations were varied

(0.5–10mMand 10–100mM, respectively) to maximize the formation of closed

or compact nucleotide-bound protein complexes.

Negative-stained samples were imaged using a Tecnai T20 and G2 Spirit

TEMs (FEI) operated at 120 keV. Data used in the final 3D reconstructions

were collected from a single source. Micrograph images were recorded using

a 4k 3 4k CCD camera (Gatan) at 67,0003 and 68,0003 magnification with

2.25 and 2.21 A pixel size for the T20 and T12 microscopes, respectively.

Three-Dimensional Reconstructions and AnalysisThe 3D reconstructions were performed using EMAN (Ludtke et al., 1999). For

the HtpG:AMPPNP and HtpG:ADP reconstructions a subset of class averages

were chosen and used to generate an initial model by the cross common lines

method (Figure S4). For HtpG:AMPPNP some preferential orientation of the

side views was observed; therefore, a 60! tilted data set was also collected

and added to the 0! data in the refinement. For the Hsc82:AMPPNP recon-

struction, the HtpG:AMPPNP initial model was used. Defocus values ranged

between 1.2 and 1.5 mM defocus, calculated by ctfit. The reconstructions in-

volved 10 (HtpG:ADP) and 12 (HtpG:AMPPNP and Hsc82:AMPPNP) rounds

of refinement and the final models were low-pass filtered to 20 A. A total of

5778 (4421 for the final model), 5035 (3812 for the final model) and 7549

(6197 for the final model) particles were used in the HtpG:AMPPNP,

Figure 7. Species-Dependent Conforma-tional Equilibrium Model for the Hsp90Chaperone CycleThe Hsp90 chaperone cycle is represented as

a dynamic conformational equilibrium where ATP

stabilizes but does not completely determine the

closed conformation. The horizontal bars repre-

sent the different fractions of open (light color)

and closed (dark color) conformations for E. coli

(green), yeast (blue), and human (orange) Hsp90

in the apo form (1) or bound to ATP (2) or ADP

(3). The fraction of open and closed conformations

for E. coli and yeast were quantitatively deter-

mined (Figure 5), whereas human Hsp90 data

and the fraction of compact ADP conformations

were visually estimated.

Molecular Cell

Conserved States of the Hsp90 Molecular Chaperone

638 Molecular Cell 32, 631–640, December 5, 2008 ª2008 Elsevier Inc.

HtpG:ADP, and Hsc82:AMPPNP reconstructions, respectively. During early

rounds of refinement, large ‘‘classkeep’’ (1.0) and ‘‘classiter’’ (8) values were

used to reduce initial model bias. These values were made more stringent in

later rounds to improve resolution. For visualization, the maps were contoured

to the approximate molecular weight of HtpG (142 kDa) or Hsc82 (162 kDa).

The resolution of the final maps was measured by the even-odd test in

EMAN where the Fourier shell correlation method is used to compare two

models generated from a random split of the data. The resolution was set to

the spatial frequency at an FSC value equal to 0.5 (Figure S2).

Reconstructions were compared with crystal structure data by rigid body

docking of the structure maps using the Chimera ‘‘fit model in map’’ command

(Pettersen et al., 2004). The fit of the yeast Hsp90:AMPPNP structure and the

model docked by rigid body domain fitting were compared by calculating

a cross-correlation value using the Laplacian filter in the Situs software package

(ChaconandWriggers,2002).Differencemaps (Figure6A)werecalculatedusing

SPIDER (Frank et al., 1996) by generating a 3D volume (low-pass filtered to 20 A)

of the Hsp82:AMPPNP crystal structure model aligned to the Hsc82:AMPPNP

map. The aligned volumes were subtracted and the resulting difference maps

were contoured to equivalent molecular weight values for visualization. Rigid

body domain fitting of the Hsp82:AMPPNP structure was achieved by a combi-

nation of a 3D grid search in angle space and simultaneous least-squares opti-

mization of rotations and translations using custom-developed software.

Conformational Matching by 2D Multireference AlignmentWe used a multireference method to identify the fraction of open and closed

conformations under different conditions. Three independent data sets (500–

800 single particles for each) of apo HtgG, HtpG:AMPPNP, apo Hsc82 and

Hsc82:AMPPNP were first collected. For the references, 2D projections

were generated from 3D volumes of the closed yeast Hsp90:AMPPNP struc-

ture and an extended apo-HtgG model derived from solution SAXS studies

(Krukenberg et al., 2008). The reference projections of the closed and ex-

tended conformations were first aligned to each other to determine the top dis-

tinct views by correlation value (0.7 cutoff). The single-particle data sets were

then aligned to the combined set references and the fraction that matched the

distinct closed or extended references was determined under the given condi-

tions. Particles that aligned to top-down views of the closed state were ex-

cluded due to similarities with the compact conformation.

Crosslinking and 2D Analysis of Human Hsp90aand Yeast Hsc82 ProteinsHsp90 proteins were incubated at 150 nM as discussed above, except sam-

ples were diluted into a buffer containing 20 mM HEPES (pH 7.5). After a

20 min equilibration step, glutaraldehyde was added to 0.005% and samples

were then incubated for 10 min. The crosslinking reaction was quenched with

20 mM Tris (pH 7.5). Samples were then negatively stained as discussed

above. Single-particle data sets of approximately 1000 were collected for

each and reference-free class averages were then generated (EMAN), and

aligned to 2D projections of the appropriate reconstructions for comparison.

SUPPLEMENTAL DATA

The Supplemental Data include eight figures and are available at http://www.

cell.com/molecular-cell/supplemental/S1097-2765(08)00766-1.

ACKNOWLEDGMENTS

Funding for this project was provided by the Howard HughesMedical Institute,

University of California Discovery Grant bio03-10401, and an American Cancer

Society postdoctoral fellowship. Special thanks to U. Boettcher, Y. Chang,

C. Cunningham, J. Kollman, K. Krukenberg, T. Street, L. Lavery, and L. Rice

for many helpful discussions and comments on the manuscript.

Received: June 7, 2008

Revised: September 16, 2008

Accepted: October 23, 2008

Published: December 4, 2008

REFERENCES

Ali, M.M., Roe, S.M., Vaughan, C.K., Meyer, P., Panaretou, B., Piper, P.W.,

Prodromou, C., and Pearl, L.H. (2006). Crystal structure of an Hsp90-nucleo-

tide-p23/Sba1 closed chaperone complex. Nature 440, 1013–1017.

Ban, C., Junop, M., and Yang, W. (1999). Transformation of MutL by ATP bind-

ing and hydrolysis: a switch in DNA mismatch repair. Cell 97, 85–97.

Bron, P., Giudice, E., Rolland, J.P., Buey, R.M., Barbier, P., Diaz, J.F., Peyrot,

V., Thomas, D., and Garnier, C. (2008). Apo-Hsp90 coexists in two open

conformational states in solution. Biol. Cell 100, 413–425.

Bukau, B., and Horwich, A.L. (1998). The Hsp70 and Hsp60 chaperone

machines. Cell 92, 351–366.

Chacon, P., and Wriggers, W. (2002). Multi-resolution contour-based fitting of

macromolecular structures. J. Mol. Biol. 317, 375–384.

Chadli, A., Ladjimi, M.M., Baulieu, E.E., and Catelli, M.G. (1999). Heat-induced

oligomerization of the molecular chaperone Hsp90. Inhibition by ATP and

geldanamycin and activation by transition metal oxyanions. J. Biol. Chem.

274, 4133–4139.

Chu, F., Maynard, J.C., Chiosis, G., Nicchitta, C.V., and Burlingame, A.L.

(2006). Identification of novel quaternary domain interactions in the Hsp90

chaperone, GRP94. Protein Sci. 15, 1260–1269.

Cunningham, C.N., Krukenberg, K.A., and Agard, D.A. (2008). Intra- and inter-

monomer interactions are required to synergistically facilitate ATP hydrolysis

in Hsp90. J. Biol. Chem. 283, 21170–21178.

Dollins, D.E., Warren, J.J., Immormino, R.M., and Gewirth, D.T. (2007). Struc-

tures of GRP94-nucleotide complexes reveal mechanistic differences

between the hsp90 chaperones. Mol. Cell 28, 41–56.

Dutta, R., and Inouye, M. (2000). GHKL, an emergent ATPase/kinase super-

family. Trends Biochem. Sci. 25, 24–28.

Felts, S.J., and Toft, D.O. (2003). p23, a simple protein with complex activities.

Cell Stress Chaperones 8, 108–113.

Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., and Leith,

A. (1996). SPIDER and WEB: processing and visualization of images in 3D

electron microscopy and related fields. J. Struct. Biol. 116, 190–199.

Frey, S., Leskovar, A., Reinstein, J., and Buchner, J. (2007). The ATPase cycle

of the endoplasmic chaperone Grp94. J. Biol. Chem. 282, 35612–35620.

Garcia-Cardena, G., Fan, R., Shah, V., Sorrentino, R., Cirino, G., Papapetro-

poulos, A., and Sessa,W.C. (1998). Dynamic activation of endothelial nitric ox-

ide synthase by Hsp90. Nature 392, 821–824.

Harris, S.F., Shiau, A.K., and Agard, D.A. (2004). The crystal structure of the

carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, re-

veals a potential substrate binding site. Structure 12, 1087–1097.

Hartl, F.U., and Hayer-Hartl, M. (2002). Molecular chaperones in the cytosol:

from nascent chain to folded protein. Science 295, 1852–1858.

Holt, S.E., Aisner, D.L., Baur, J., Tesmer, V.M., Dy, M., Ouellette, M., Trager,

J.B., Morin, G.B., Toft, D.O., Shay, J.W., et al. (1999). Functional requirement

of p23 and Hsp90 in telomerase complexes. Genes Dev. 13, 817–826.

Johnson, B.D., Schumacher, R.J., Ross, E.D., and Toft, D.O. (1998). Hop

modulates Hsp70/Hsp90 interactions in protein folding. J. Biol. Chem. 273,

3679–3686.

Krukenberg, K.A., Forster, F., Rice, L.M., Sali, A., and Agard, D.A. (2008).

Multiple Conformations of E. coli Hsp90 in Solution: Insights into the Confor-

mational Dynamics of Hsp90. Structure 16, 755–765.

Ludtke, S.J., Baldwin, P.R., and Chiu, W. (1999). EMAN: semiautomated soft-

ware for high-resolution single-particle reconstructions. J. Struct. Biol. 128,

82–97.

Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A.L., and Hartl, F.U.

(1991). Chaperonin-mediated protein folding at the surface of groEL through

a ‘molten globule’-like intermediate. Nature 352, 36–42.

McLaughlin, S.H., Smith, H.W., and Jackson, S.E. (2002). Stimulation of the

weak ATPase activity of human hsp90 by a client protein. J. Mol. Biol. 315,

787–798.

Molecular Cell

Conserved States of the Hsp90 Molecular Chaperone

Molecular Cell 32, 631–640, December 5, 2008 ª2008 Elsevier Inc. 639

McLaughlin, S.H., Sobott, F., Yao, Z.P., Zhang, W., Nielsen, P.R., Grossmann,

J.G., Laue, E.D., Robinson, C.V., and Jackson, S.E. (2006). The co-chaperone

p23 arrests the Hsp90 ATPase cycle to trap client proteins. J. Mol. Biol. 356,

746–758.

McLaughlin, S.H., Ventouras, L.A., Lobbezoo, B., and Jackson, S.E. (2004). In-

dependent ATPase activity of Hsp90 subunits creates a flexible assembly plat-

form. J. Mol. Biol. 344, 813–826.

Meyer, P., Prodromou, C., Hu, B., Vaughan, C., Roe, S.M., Panaretou, B.,

Piper, P.W., and Pearl, L.H. (2003). Structural and functional analysis of the

middle segment of hsp90: implications for ATP hydrolysis and client protein

and cochaperone interactions. Mol. Cell 11, 647–658.

Meyer, P., Prodromou, C., Liao, C., Hu, B., Mark Roe, S., Vaughan, C.K.,

Vlasic, I., Panaretou, B., Piper, P.W., and Pearl, L.H. (2004). Structural basis

for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machin-

ery. EMBO J. 23, 511–519.

Nathan, D.F., Vos, M.H., and Lindquist, S. (1997). In vivo functions of the Sac-

charomyces cerevisiae Hsp90 chaperone. Proc. Natl. Acad. Sci. USA 94,

12949–12956.

Neckers, L. (2002). Hsp90 inhibitors as novel cancer chemotherapeutic

agents. Trends Mol. Med. 8, S55–S61.

Ohi, M., Li, Y., Cheng, Y., and Walz, T. (2004). Negative staining and image

classification—powerful tools in modern electron microscopy. Biol. Proced.

Online 6, 23–34.

Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M.,

Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera–a visualization system

for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612.

Picard, D. (2002). Heat-shock protein 90, a chaperone for folding and regula-

tion. Cell. Mol. Life Sci. 59, 1640–1648.

Picard, D., Khursheed, B., Garabedian, M.J., Fortin, M.G., Lindquist, S., and

Yamamoto, K.R. (1990). Reduced levels of hsp90 compromise steroid recep-

tor action in vivo. Nature 348, 166–168.

Pratt, W.B., Galigniana, M.D., Morishima, Y., and Murphy, P.J. (2004). Role of

molecular chaperones in steroid receptor action. Essays Biochem. 40, 41–58.

Prodromou, C., Panaretou, B., Chohan, S., Siligardi, G., O’Brien, R., Ladbury,

J.E., Roe, S.M., Piper, P.W., and Pearl, L.H. (2000). The ATPase cycle of Hsp90

drives a molecular ‘clamp’ via transient dimerization of the N-terminal do-

mains. EMBO J. 19, 4383–4392.

Prodromou, C., Roe, S.M., O’Brien, R., Ladbury, J.E., Piper, P.W., and Pearl,

L.H. (1997). Identification and structural characterization of the ATP/ADP-

binding site in the Hsp90 molecular chaperone. Cell 90, 65–75.

Richter, K., and Buchner, J. (2001). Hsp90: chaperoning signal transduction.

J. Cell. Physiol. 188, 281–290.

Richter, K., Soroka, J., Skalniak, L., Leskovar, A., Hessling, M., Reinstein, J.,

and Buchner, J. (2008). Conserved conformational changes in the ATPase

cycle of human Hsp90. J. Biol. Chem. 283, 17757–17765.

Roe, S.M., Prodromou, C., O’Brien, R., Ladbury, J.E., Piper, P.W., and Pearl,

L.H. (1999). Structural basis for inhibition of the Hsp90 molecular chaperone

by the antitumor antibiotics radicicol and geldanamycin. J. Med. Chem. 42,

260–266.

Sato, S., Fujita, N., and Tsuruo, T. (2000). Modulation of Akt kinase activity by

binding to Hsp90. Proc. Natl. Acad. Sci. USA 97, 10832–10837.

Shiau, A.K., Harris, S.F., Southworth, D.R., and Agard, D.A. (2006). Structural

Analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conforma-

tional rearrangements. Cell 127, 329–340.

Vaughan, C.K., Gohlke, U., Sobott, F., Good, V.M., Ali, M.M., Prodromou, C.,

Robinson, C.V., Saibil, H.R., and Pearl, L.H. (2006). Structure of an Hsp90-

Cdc37-Cdk4 complex. Mol. Cell 23, 697–707.

Young, J.C., Agashe, V.R., Siegers, K., and Hartl, F.U. (2004). Pathways of

chaperone-mediated protein folding in the cytosol. Nat. Rev. Mol. Cell Biol.

5, 781–791.

Zhang, W., Hirshberg, M., McLaughlin, S.H., Lazar, G.A., Grossmann, J.G.,

Nielsen, P.R., Sobott, F., Robinson, C.V., Jackson, S.E., and Laue, E.D.

(2004). Biochemical and structural studies of the interaction of Cdc37 with

Hsp90. J. Mol. Biol. 340, 891–907.

Zhao, R., Davey, M., Hsu, Y.C., Kaplanek, P., Tong, A., Parsons, A.B., Krogan,

N., Cagney, G., Mai, D., Greenblatt, J., et al. (2005). Navigating the chaperone

network: an integrative map of physical and genetic interactions mediated by

the hsp90 chaperone. Cell 120, 715–727.

Zhu, X., Zhao, X., Burkholder, W.F., Gragerov, A., Ogata, C.M., Gottesman,

M.E., and Hendrickson, W.A. (1996). Structural analysis of substrate binding

by the molecular chaperone DnaK. Science 272, 1606–1614.

Molecular Cell

Conserved States of the Hsp90 Molecular Chaperone

640 Molecular Cell 32, 631–640, December 5, 2008 ª2008 Elsevier Inc.