Mitochondrial Hsp90 is a ligand-activated molecularchaperone coupling ATP binding to dimer closurethrough a coiled-coil intermediateNuri Sunga, Jungsoon Leea, Ji-Hyun Kima,1, Changsoo Changb, Andrzej Joachimiakb, Sukyeong Leea,2,and Francis T. F. Tsaia,c,d,2

aVerna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030; bStructural Biology Center,Biosciences Division, Argonne National Laboratory, Argonne, IL 60439; cDepartment of Molecular and Cellular Biology, Baylor College of Medicine,Houston, TX 77030; and dDepartment of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030

Edited by Manu Sharma, Weill Cornell Medical College, New York, NY, and accepted by the Editorial Board January 28, 2016 (received for reviewAugust 14, 2015)

Heat-shock protein of 90 kDa (Hsp90) is an essential molecularchaperone that adopts different 3D structures associated withdistinct nucleotide states: a wide-open, V-shaped dimer in theapo state and a twisted, N-terminally closed dimer with ATP.Although the N domain is known to mediate ATP binding, howHsp90 senses the bound nucleotide and facilitates dimer closureremains unclear. Here we present atomic structures of humanmitochondrial Hsp90N (TRAP1N) and a composite model of intactTRAP1 revealing a previously unobserved coiled-coil dimer con-formation that may precede dimer closure and is conserved inintact TRAP1 in solution. Our structure suggests that TRAP1 nor-mally exists in an autoinhibited state with the ATP lid bound tothe nucleotide-binding pocket. ATP binding displaces the ATP lidthat signals the cis-bound ATP status to the neighboring subunitin a highly cooperative manner compatible with the coiled-coilintermediate state. We propose that TRAP1 is a ligand-activatedmolecular chaperone, which couples ATP binding to dramaticchanges in local structure required for protein folding.

TRAP1 | Hsp90 | molecular chaperone

Heat-shock protein of 90 kDa (Hsp90) is a conserved ATP-dependent molecular chaperone (1–4), which together with

heat-shock protein of 70 kDa (Hsp70) (5–7) and a cohort ofcochaperones (8–10), promotes the late-stage folding of Hsp90client proteins (11). It is presumed that almost 400 differentproteins, including a majority of signaling and tumor promotingproteins, depend on cytosolic Hsp90 for folding (12). Conse-quently, the ability to inactivate multiple oncogenic pathwayssimultaneously has made Hsp90 a major target for drug de-velopment (13), with several Hsp90 inhibitors currently un-dergoing clinical trials (14).Hsp90 chaperones display conformational plasticity in solution

(2, 15, 16), with different adenine nucleotides either facilitatingor stabilizing distinct Hsp90 dimer conformations (17–19). In-terestingly, apo Hsp90 forms a wide-open, V-shaped dimerwith the N domains separated by as much as 101 Å (18). Thisopen conformation is markedly distinct from the intertwined,N-terminally closed dimer with ATP bound (20, 21). Becausethe open-state dimer cannot signal the nucleotide status betweenneighboring subunits, an intermediate conformation precedingdimer closure must exist, which so far has remained elusive.Apart from cytosolic Hsp90s, Hsp90 homologs are found in the

endoplasmic reticulum, chloroplasts, and mitochondria (Fig. S1) (22).The tumor necrosis factor receptor-associated protein 1 (TRAP1) isthe mitochondrial Hsp90 paralog, which prevents apoptosis andprotects mitochondria against oxidative damage (23–25). TRAP1 iswidely expressed in many tumors (24, 26, 27), but not in mitochon-dria of most normal tissues (24), benign prostatic hyperplasia (26), orhighly proliferating, nontransformed cells (27). Notably, it was foundthat TRAP1 not only promotes neoplastic growth, but also conferstumorigenic potential on nontransformed cells (27), indicating a

major role of TRAP1 in tumorigenesis, although TRAP1’s specificfunction remains poorly understood (28).TRAP1 is a multidomain protein consisting of an N-terminal or N

domain (TRAP1N), a middle domain (TRAP1M), and a C-terminalor C domain (TRAP1C), but it lacks the charged linker foundin eukaryotic Hsp90 paralogs. Human TRAP1 is preceded by amitochondrial localization sequence (MLS) of 59 residues thatare cleaved off during import (29). The mature form of TRAP1 isa homodimer held together by TRAP1C, with a second, ATP binding-dependent dimer interface in TRAP1N. The crystal structures ofzebrafish TRAP1 (zTRAP1) and zebrafish and human TRAP1NMbound to ADPNP were recently reported (21, 30), and are largelyconsistent with our current understanding of Hsp90 chaperones.In the ATP-bound state, the N-terminal extension (known as theN strap) straddles the N domain of the neighboring subunit,thereby stabilizing the structure of the closed-state dimer (21, 30).The ordered segment of the N strap significantly lengthens thepreviously observed β-strand swap and may function as a regula-tory element that controls TRAP1 function (16, 21). Interestingly,intact zTRAP1-ADPNP crystallized as an asymmetric dimer thatcould support a sequential ATP hydrolysis mechanism (31, 32);

Significance

Mitochondrial heat-shock protein of 90 kDa (Hsp90) (TRAP1)promotes cell survival and is essential for neoplastic growth.Exploiting human TRAP1 for drug development requires de-tailed structural andmechanistic understanding. Whereas TRAP1adopts different conformations associated with distinct nucleo-tide states, how the TRAP1 dimer senses the bound nucleotideand signals this information to the neighboring subunit remainsunknown. We show that unliganded TRAP1 forms a previouslyunobserved coiled-coil dimer and is found in an autoinhibitedstate. ATP binding in cis displaces the ATP lid that signals thenucleotide status to the trans subunit. Our findings suggest thathuman TRAP1 is a ligand-activated molecular chaperone, whichcouples ATP binding to local changes in structure facilitatingdimer closure needed for protein folding.

Author contributions: N.S., J.L., S.L., and F.T.F.T. designed research; N.S., J.L., J.-H.K., C.C.,and S.L. performed research; N.S., J.L., J.-H.K., C.C., A.J., S.L., and F.T.F.T. contributed newreagents/analytic tools; N.S., J.L., C.C., A.J., S.L., and F.T.F.T. analyzed data; and N.S., J.L.,J.-H.K., C.C., A.J., S.L., and F.T.F.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. M.S. is a guest editor invited by the EditorialBoard.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 5F5R and 5F3K).1Present address: Pennington Biomedical Research Center, Louisiana State University,Baton Rouge, LA 70808.

2To whom correspondence may be addressed. Email: slee@bcm.edu or ftsai@bcm.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1516167113/-/DCSupplemental.

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however, no asymmetric nucleotide binding was observed, and nomolecular contacts between cis-bound ADPNP and the N domainof the neighboring subunit were seen in TRAP1 (21, 30) and otherknown Hsp90 structures (18, 20, 33), leaving open the questionof how TRAP1 senses and signals the nucleotide-bound statusbetween subunits.Here we present atomic structures of human TRAP1N

(hTRAP1N) alone and in complex with ADPNP. Unexpectedly, wefound that unliganded hTRAP1N forms a previously unobservedcoiled-coil dimer that is distinct from the proposed open-state andclosed-state conformations (16, 21, 30). Importantly, intact hTRAP1forms a similar coiled-coil dimer in solution, but only in the absenceof ATP. Our findings show that ATP binding triggers a dramaticchange in local structure and displaces the ATP lid, which is boundto the ATP-binding pocket, indicating that TRAP1 normally exists inan autoinhibited state. Strikingly, mutations of conserved residuesthat impair lid binding stimulate the hTRAP1 ATPase activity in ahighly cooperative manner, supporting a previously unknown role ofthe ATP lid in signaling the cis-bound nucleotide status to the transsubunit, which is compatible with the coiled-coil dimer. Finally, wedemonstrate that TRAP1 folding requires ATP and the functionalcooperation of the mitochondrial Hsp70 chaperone system, sup-porting the existence of a mitochondrial Hsp90-Hsp70 supercomplexthat may present a new target for drug development.

ResultsAtomic Structure of Monomeric hTRAP1N in the ATP State. Theemerging role of TRAP1 as a potent drug target necessitatesatomic structure information of the human protein to exploit thisinformation to develop mitochondrial Hsp90 inhibitors. The 3.3-Åcrystal structure of a closed-state human TRAP1NM (hTRAP1NM)dimer bound to ADPNP, which shares close overall similarity withthe previously determined crystal structure of the zTRAP1NM-ADPNP dimer (21), was reported recently (30). Conversely,hTRAP1N-ADPNP crystallized as a monomer (Fig. 1A andFig. S2A), with crystals diffracting to 1.85-Å resolution (Table S1).The absence of a dimer is not the result of TRAP1 truncation,however, given that TRAP1N-ADPNP can also crystallize as anintertwined N-domain dimer (Fig. S3) indistinguishable from thatobserved with intact zTRAP1-ADPNP (21). The 2Fo-Fc map ofmonomeric hTRAP1N is of excellent quality (Fig. S4), enablingtracing of all but the first 10 residues of the mature form ofhTRAP1N (Fig. S2A). The sole base-specific interaction betweenhTRAP1N and bound ADPNP is observed with the carboxylate side-chain of Asp158 that is evolutionary conserved (Fig. S1) and makesa hydrogen bond with the exocyclic N6-amine of adenine (Fig. 1A).Pairwise comparison of hTRAP1N and the N domain of intact

zTRAP1 (PDB ID code 4IPE) (21) shows that the two structuressuperpose with an rmsd of only 0.7 Å over 148 Cα atoms. How-ever, hTRAP1N crystallizes with the N strap bound in cis(Fig. 1A), as opposed to straddling the N domain in trans (Fig. 1B),but occupies the same binding site in the hTRAP1N monomer as

the trans segment in the ADPNP-bound TRAP1 dimer (Fig. 1C).Although this finding is surprising at first glance, the atomic struc-tures of monomeric and dimeric hTRAP1N- and hTRAP1NM-ADPNP complexes are consistent with the notion that hTRAP1-ADPNP can adopt both closed- and open-state dimers, with elevatedtemperatures favoring the closed-state conformation (16). In-terestingly, a cis-bound N-strap conformation also has beenobserved in crystal structures of hTRAP1NM-inhibitor complexes(30), suggesting that this conformation might be a common featureof monomeric TRAP1.Another notable difference is the open conformation of the ATP

lid (residues 177 to 202), which exposes the bound nucleotide tobulk solvent (Fig. 1A) as opposed to being folded over the nucle-otide-binding pocket (Fig. 1B). A similar open-lid conformation wasalso reported for monomeric Grp94N-nucleotide complexes (34),suggesting that lid closure is not critical for nucleotide binding toHsp90 chaperones in general. To determine the structural basis forlid closure, we compared the crystal structure of the hTRAP1N-ADPNP monomer with that of the zTRAP1-ADPNP dimer (21).Superposition of the two structures shows that an open-lid confor-mation is incompatible with the closed-state dimer, because it wouldsterically clash with the N domain of the neighboring subunit (Fig.1C). Thus, our structure confirms that lid closure is nonessential fornucleotide binding and is driven largely by steric interference.

Atomic Structure of hTRAP1N Reveals a Coiled-Coil Dimer. In additionto the ATP-bound state, we report the first, to our knowledge,atomic structure of unliganded hTRAP1N at 1.82-Å resolution(Table S1). Unexpectedly, the crystal structure reveals a previouslyunobserved coiled-coil dimer in the asymmetric unit of the crystal(Fig. 2A), which is markedly distinct from the proposed open- andclosed-state conformations (16, 21, 30). The coiled-coil dimer in-terface is stabilized by an extensive network of hydrogen bondsformed between the N-terminal helices from each subunit, andoccludes 1,765 Å2 of solvent accessible area. The latter is 38 Å2

more than the closed-stated zTRAP1N-ADPNP dimer (1,727 Å2)as calculated with PISA (35) over the corresponding range of res-idues (zTRAP1N residues 97 to 309), not taking into account anadditional 1,218 Å2 owing to contributions of N-strap resi-dues (zTRAP1N residues 85 to 96), which are disorderedin hTRAP1N.In addition to the previously unobserved dimer interface, it is

immediately evident that each hTRAP1N monomer adopts a moreextended, solvent-exposed conformation than previously observedADPNP-bound complexes (21, 30) (Fig. 1A). Perhaps moststrikingly, the N strap no longer forms a β-strand as seen in theADPNP-bound state, but instead forms an α-helix (Fig. 2B andFig. S2B) and pairs up with the helical N strap of the other subunitto form a zipper-like, coiled-coil dimer (Fig. 2A). The coiled-coilinterface is further stabilized by interactions between Phe183 inthe ATP lid of the trans subunit and Phe90, Glu93, and Thr94 ofthe cis subunit.

A C

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Fig. 1. Structural comparison of the hTRAP1N monomer (green) and the zTRAP1 dimer (gold). The ATP lid is in red, and the N strap is highlighted in purple.ADPNP and Asp158 (Asp173 in zTRAP1) are shown as stick models. (A and B) Crystal structures of hTRAP1N-ADPNP (A) and an equivalent N domain of zTRAP1-ADPNP with the N strap straddling the neighboring subunit (B). Only one N domain is shown. (C) Superposition of the N domains of hTRAP1N-ADPNP andzTRAP1-ADPNP showing that an extended ATP lid would sterically clash with the neighboring subunit (gray) in the closed-state dimer.

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The coiled-coil dimer structure was unexpected, given the com-mon presumption that Hsp90N is a monomer in the absence ofnucleotide (36), a conformation supported by the apo structure offull-length bacterial Hsp90 (18), which shows a wide-open, V-shapeddimer conformation. Consistently, superposing unliganded hTRAP1Nonto the equivalent domain of zTRAP1-ADPNP (21) and TRAP1homologs (18, 20, 33) shows that the helical N strap will stericallyclash with the neighboring N domain in the closed-state dimer(Fig. 2B and Fig. S5 A and B), whereas in the proposed open state,the N domains are too far apart to contact each other.To exclude the possibility that the coiled-coil dimer results

from crystal packing interactions, we determined the oligo-meric state of hTRAP1N in solution. Consistent with the crystalstructure, we found that unliganded hTRAP1N is a dimer,whereas an N-terminally truncated hTRAP1N variant lacking theN-terminal α-helix (hTRAP1NΔ107) is monomeric (Fig. 3A).It has been reported that an N-strap truncation (hTRAP1Δ84)

stimulates ATPase activity by ∼30-fold compared with the matureform of hTRAP1 (hTRAP1Δ59) (16) (Fig. 3B). Strikingly, we foundthat further deletion of the N-terminal α-helix (hTRAP1Δ107) oronly the helical N strap (hTRAP1Δ98) nearly abolished the ATPaseactivity of hTRAP1 (Fig. 3B). Our findings demonstrate that thehelical N strap has an essential regulatory function and is requiredfor formation of the coiled-coil dimer that may represent an in-termediate conformation preceding dimer closure.

The N-Terminal α-Helices of Intact hTRAP1 Form a Coiled Coil in theAbsence of Nucleotide. Our attempts to crystallize intact hTRAP1without nucleotide were unsuccessful. Thus, to generate an insilico model for hTRAP1, we examined previously reportedstructures of full-length Hsp90 chaperones to identify an Hsp90MCconformation compatible with the coiled-coil hTRAP1N dimerstructure. Because the structure of the coiled-coil intermediate hasnot been observed previously, it seemed unlikely that we wouldfind a compatible structure. Fortuitously, several crystal structureswere compatible with the imposed twofold restraint. We foundthat the X-ray structures of Grp94 in complex with ADP (PDB IDcode 2O1V) or ADPNP (PDB ID code 2O1U) (33) fit best. Thetwo structures are nearly identical to each other and superposewith an rmsd of only 0.5 Å over all Cα atoms. In our model, the Ctermini of hTRAP1N (Thr294 and Thr294′) are positioned to theirrespective N termini of Grp94MC (Leu339 and Leu339′) with anequal distance of only 4.3 Å (Fig. 3C, Top and Fig. S6).To validate our model, we wished to probe the coiled-coil

dimer interface of intact hTRAP1 in solution. Because a coiled-coil dimer is observed only in the unliganded state, we engineeredhTRAP1 variants featuring a Cys in the helical N strap, whichpotentially could form a disulfide cross-link in the absence ofnucleotide. In the crystal structure, Lys95 and Leu98 are near thecross-point of the coiled coil with Cα distances of 7.3 Å (Lys95-Lys95′) and 7.5 Å (Leu98-Leu98′), compatible with disulfide

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Fig. 3. Human TRAP1N and hTRAP1 form a coiled-coil dimer. (A) Size-exclusion chromatogram ofhTRAP1N (black curve) and hTRAP1NΔ107 (gray curve).(Inset) The hTRAP1N dimer, with the N-terminalhelices deleted in hTRAP1NΔ107 shown in gray. (B)Relative ATPase activities of hTRAP1 (Δ59) andN-terminal truncated hTRAP1 mutants lacking thefirst 84 (Δ84), 98 (Δ98), or 107 (Δ107) residues. (C) Insilico model of the intact coiled-coil hTRAP1 dimer(Top) and the crystal structure of the closed-statezTRAP1-ADPNP dimer (21) (Bottom). TRAP1N is shownin blue and magenta; TRAP1MC, in different shades ofgray. Introduced Cys sites are marked by spheres.Corresponding Cys pairs are shown in the samecolor, with distances between Cα atoms indicated.(D) Disulfide cross-linking of hTRAP1 and Cys-containinghTRAP1* variants without or with ADPNP. Cross-linkeddimers can be monomerized with 100 mM DTT (+DTT).Bands corresponding to hTRAP1 monomers (1mer) anddimers (2mer) are indicated.

A

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Fig. 2. Structural comparison of the coiled-coil hTRAP1N dimer (blue/magenta) and the closed-state zTRAP1-ADPNP dimer (gold/pink). (A) Crystal structureof the unliganded hTRAP1N dimer. Phe183 is shown as CPK model. (B) Superposition of unliganded hTRAP1N (blue) onto the structure of the zTRAP1-ADPNPdimer, illustrating the local structural rearrangements in hTRAP1N on nucleotide binding. (1) ATP binding displaces the ATP lid from the nucleotide-bindingpocket, concomitant with (2) the N strap undergoing a structural transition from an α-helix to a β-strand that straddles the neighboring subunit and stabilizesthe closed-state dimer. (3) Owing to steric interference, the ATP lid must fold over the nucleotide-binding pocket trapping the bound nucleotide.

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bond formation (Fig. 3C, Top), as opposed to 17.1 Å and 15.4 Åin the ADPNP-bound state (21) (Fig. 3C, Bottom). Thus, wegenerated hTRAP1 mutants by replacing Lys95 or Leu98 withCys in a Cys-free hTRAP1 variant (hTRAP1*), which is fullyfunctional (Fig. S7). Notably, hTRAP1*K95C and hTRAP1*L98Cformed a cross-linked dimer only in the absence of nucleotide(Fig. 3D, lanes 6 and 7), but not in the presence of ADPNP (Fig.3D, lanes 9 and 10). The cross-linking reaction is reversible withDTT (Fig. 3D, compare lanes 6 and 7 with lanes 3 and 4), and nocross-linked products were observed with hTRAP1 (Fig. 3D, lane1), underscoring the specificity of the cross-linking reaction.Taken together, our findings lead us to conclude that unligandedhTRAP1 adopts a similar coiled-coil dimer in solution as ob-served with hTRAP1N in the crystal.

The ATP Lid Functions as a Key Regulatory Element. The crystalstructure of TRAP1 confirmed that the nucleotide-binding do-main shares the canonical Bergerat fold (37), which featuresseveral conserved motifs, including the G1 box and G2 box thatflank the ATP lid (Fig. S1). It was initially proposed that the lidrepresents an ATP-binding motif (37) that folds over thenucleotide-binding pocket to stabilize bound ATP in GHKL (gyrase,Hsp90, histidine kinase, MutL) ATPases (38, 39). Although de-letion of the lid did not affect nucleotide binding to Hsp90,lidless yeast Hsp90 lacks ATPase activity and cannot rescue anhsp82−/hsc82− yeast strain (40), underscoring the importance ofthe ATP lid to Hsp90 function.In the crystal structure of hTRAP1N-ADPNP, the ATP lid is

folded away from the nucleotide-binding pocket, consistent with thenotion that the lid is dispensable for ATP binding. Interestingly, inthe crystal structure of unliganded hTRAP1N, the ATP lid foldsover the nucleotide-binding pocket. Closer inspection reveals thatGln200, Phe201, and Gly202 of the G2 box overlapped with theribose ring and the α-phosphate of the bound nucleotide (Fig. 4A),effectively competing with nucleotide binding (Fig. S8A). A similarG2 box motif interaction was also observed in the crystal structureof full-length bacterial Hsp90 in the apo conformation (Fig. S8B)(18), indicating that mitochondrial and bacterial Hsp90 may nor-mally exist in an autoinhibited state. Interestingly, in hTRAP1, theamine group of the Gln200 side chain superposed with the exocyclic

N6-amine of adenine in the ADPNP-bound state, and formed ahydrogen bond with the carboxylate side chain of Asp158 (Fig. 4A).Because the ATP lid has been implicated in the regulation of

Hsp90 ATPase activity (40, 41), we reasoned that the conservedG2 box motif might function as a sensor of nucleotide binding,providing a means to signal the cis-bound nucleotide status to theneighboring subunit. Consistent with a coordinated ATP hy-drolysis mechanism (21, 31), hTRAP1 variants impaired in lidbinding should stimulate ATPase activity by signaling a consti-tutive cis-bound nucleotide status to the trans subunit.To test this idea, we mutated Asp158, Gln200, and Phe201 either

alone or in combination. We found that our engineered hTRAP1variants retained ATPase activity (Fig. 4 B and C), including anhTRAP1 variant featuring a mutation of Asp158 to Asn. Althoughin principle, an Asn is compatible with ATP binding, our findingdiffers from previous observations with cytosolic Hsp90 (42, 43).Surprisingly, hTRAP1Q200A/F201A showed only 4.1-fold higherATPase activity than hTRAP1, slightly less than that observed withhTRAP1F201A alone (Fig. 4B), indicating that lid binding is drivenlargely by van der Waals interactions. Interestingly, we found that theATPase activity of TRAP1D158N was increased only slightly (2.5-fold),whereas a mutation of Asp158 to Ala stimulated ATPase activity by14.6-fold (Fig. 4C), suggesting that the stimulated ATPase activity ofhTRAP1 variants depends on the nature of the introduced substitution.The greatest stimulation was observed with hTRAP1D158A/F201A,which increased ATPase activity by 33.0-fold, significantly greaterthan the increases seen with hTRAP1D158A/Q200A/F201A triplemutant (18.1-fold) or any of the single mutants alone (Fig. 4C).To determine whether the stimulation of ATPase activity

resulted from cooperative interactions between hTRAP1 subunits,we generated heterodimers of hTRAP1 and hTRAP1D158A and ofhTRAP1 and hTRAP1D158A/F201A by mixing wild-type and mutantsubunits in defined molar ratios, keeping the total protein amountconstant. We would expect to observe a linear relationship if thestimulated ATPase activity resulted from an increase in activitywithin one subunit, and a nonlinear relationship if stimulationoccurred between neighboring subunits. As shown in Fig. 4D, weobserved a clear nonlinear relationship with both the hTRAP1/hTRAP1D158A and hTRAP1/hTRAP1D158A/F201A heterodimers.Thus, mutations that impair lid binding in cis stimulate theATPase activity in trans. Taken together, our findings suggest thatthe ATP lid competes with nucleotide binding and is displaced byATP, providing a means to signal the cis-bound nucleotide statusto the neighboring subunit that must be in close physical prox-imity, a conformation that is compatible with the coiled-coil dimer.

TRAP1 Cooperates with Mitochondrial Chaperones in Folding. Todetermine the significance of the unliganded and ATP-boundTRAP1 conformation, we developed an assay to monitor the ATP-dependent hTRAP1 chaperone activity. It is known that cytosolicHsp90 requires Hsp70 and a cohort of cochaperones to promote

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Fig. 4. Conserved residues of the ATP-lid G2 box motif compete with ATPfor binding to the ATP-binding pocket. (A) Superposition of unliganded andADPNP-bound hTRAP1N. Only ADPNP of the hTRAP1N-ADPNP complex isshown. (B and C) ATPase rates, expressed in μmol ATP hydrolyzed per minper μmol protein of hTRAP1 and hTRAP1 variants carrying a mutation inGln200 or Phe201 (B) and/or Asp158 (C). Averages of three independentmeasurements ± SD are shown. (D) Relative ATPase activities of mixtures ofhTRAP1 and hTRAP1D158A (red), and hTRAP1 and hTRAP1D158A/F201A heter-odimers (cyan) at indicated ratios. The dashed line represents the theoreti-cal, linear relationship if the stimulated ATPase activities resulted from anincrease in ATPase activity in only one subunit.

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Fig. 5. TRAP1 requires the mitochondrial Hsp70 system for protein folding.Averages of three independent measurements ± SD are shown. (A) Reac-tivation of heat-denatured FFL after 120 min by hHsp90, hTRAP1, or bHsp90together with RRL or the bacterial Hsp70 system (KJE). Recovered FFL ac-tivities are expressed relative to native FFL. (B) Reactivation of heat-dena-tured FFL without hTRAP1 (no TRAP1), with hTRAP1 (TRAP1), or withhTRAP1 and 5 mM ATP (TRAP1-ATP), and together with Mortalin/Mdj1/Mge1. FFL recovery was monitored every 20 min for 120 min.

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the folding of Hsp90 clients (1, 10). Interestingly, hTRAP1 cannotfunctionally replace cytosolic Hsp90 in vitro (29, 44), suggesting theneed for other factors to reconstitute TRAP1-dependent folding.Rabbit reticulocyte lysate (RRL) provides a rich source of pro-

teins, and can substitute for Hsp70 and essential cochaperones toreconstitute active folding by cytosolic Hsp90 in vitro (45). AlthoughRRL facilitated hHsp90-dependent folding of heat-denaturedfirefly luciferase (FFL), no recovered FFL activity was observedwhen hHsp90 was replaced with either hTRAP1 or bacterial Hsp90(Fig. 5A). However, bacterial Hsp90 could recover FFL activitywhen RRL was replaced with the bacterial Hsp70 system (Fig. 5A).Although perplexing on first sight, it has been reported that

RRLs mostly lack functional mitochondria (46). Thus, we rea-soned that our inability to reconstitute TRAP1-dependent proteinfolding with RRL might be due to the lack of one or more com-ponents of the mitochondrial chaperone system. Indeed, we foundthat hTRAP1 reactivated heat-denatured FFL when RRL wasreplaced with the mitochondrial Hsp70 system (Fig. 5B). Impor-tantly, hTRAP1’s ability to reactivate FFL requires ATP duringheat denaturation, whereas the presence of unliganded hTRAP1and the absence of hTRAP1 resulted in very low and no recoveredFFL activity, respectively (Fig. 5B).

DiscussionOur findings provide mechanistic insight into how ATP-binding–induced local changes in TRAP1 structure facilitate dimer closure,and suggest that TRAP1 is a ligand (ATP)-activated molecularchaperone. Although our results reconcile disparate structural andbiochemical observations, they also reveal nuances specific to mi-tochondrial Hsp90. For instance, we find that an hTRAP1 variantfeaturing an Asp158-to-Asn mutation behaves differently from thatreported for cytosolic Hsp90s (42, 43). Although an Asn in place ofan Asp is not unprecedented among GHKL ATPases (47), it in-dicates that the hTRAP1 ATPase activity is fine-tuned differentlyfrom other Hsp90s. Moreover, the inability to functionally interactwith known Hsp90 cochaperones suggests that TRAP1 is de-pendent on other regulatory elements integral to TRAP1, such asthe N strap (16) and the ATP lid, whose function is uncovered here.Our findings suggest that TRAP1-ATP can in principle adopt

two distinct conformations: a closed-state dimer (ATP closed)with the N strap swapped between subunits (21, 30) and an open-state dimer (ATP open) with the N strap bound in cis (Fig. 6),which is consistent with the observed conformational plasticity ofHsp90 in solution (2, 15, 16). However, we propose that the ATPopen state does not promote protein folding.More importantly, our findings support a role of the ATP lid as a

sensor of nucleotide binding. ATP binding in cis displaces the ATPlid that in turn signals the nucleotide status to the trans subunit. Todo so, the N domains must be in close physical proximity, aconformation that is fully compatible with our coiled-coil in-termediate dimer. The role of the Gln200 side chain is perhapsmost intriguing. In the crystal structure of unliganded hTRAP1N,

the Gln200 side chain mimics the bound nucleotide and con-tributes toward lid binding by forming a hydrogen bond with theAsp158 side chain (Fig. 4A). Although an inhibitory lid confor-mation also has been observed with full-length bacterial Hsp90(18), the interaction between the conserved Gln200 and Asp158side chains is new. Consistent with a nucleotide sensor function,hTRAP1 variants carrying a mutation in the conserved Asp158or Phe201, which impairs lid-binding, show highly stimulatedATPase activity (Fig. 4 B and C). On the other hand, substitutingGln200 with Ala has a negligible effect on ATPase activity, andhTRAP1 variants carrying an additional mutation in Asp158,Phe201, or both exhibit similar or reduced ATPase activity com-pared with those that do not feature the Gln200 mutation (Fig. 4 Band C). Taken together, these findings point to an additional rolefor Gln200 in the ATPase cycle. In the crystal structure ofzTRAP1-ADPNP (PDB ID code 4IPE-A) (21), the equivalentglutamine forms water-mediated interactions with the main chainof a β-bridge featuring the ATP sensor (Arg402) (48). Thus, wespeculate that, in addition to lid binding, Gln200 is needed toposition the ATP-sensor loop required for full ATPase activity.Finally, we have demonstrated that TRAP1-dependent protein

folding requires ATP (Fig. 5B). ATP hydrolysis may be needed forsubstrate release, as has been reported for cytosolic Hsp90 (49). Inaddition to ATP, TRAP1-dependent protein folding also requiresthe functional cooperation of the mitochondrial Hsp70 system.The latter supports a direct physical interaction, as was recentlyreported for cytosolic Hsp90 chaperones (5–7, 50), and opens upnew avenues for drug development by targeting the specific in-teraction between mitochondrial chaperones.

Experimental ProceduresCloning, mutagenesis, and protein expression and purification proceduresare described in SI Experimental Procedures.

Crystallization. Human TRAP1N crystals were grown at 14 °C from hangingdrops. Then 2 μL of hTRAP1N (25 mg/mL) in 30 mM Tris·HCl pH 8.5, 0.15 MNaCl, and 1 mM tris(2-carboxyethyl)phosphine (TCEP) was mixed with anequal volume of reservoir solution consisting of 100 mM Hepes pH 7.5,100 mM CaCl2, 23% (wt/vol) PEG 3350, 4% (vol/vol) isopropanol, and 0.4 μLof 1 M LiCl. hTRAP1N crystals were harvested in reservoir solution containing5% (vol/vol) PEG400, but reducing the PEG 3350 concentration to 21%(wt/vol), and flash frozen in liquid nitrogen. hTRAP1N-ADPNP was preparedby incubating hTRAP1N (55 mg/mL) in 50 mM Tris·HCl pH 7.6, 0.2M NaCl, and1 mM TCEP with 5 mM ADPNP and 10mMMgCl2 for 30 min on ice. The samplewas mixed with an equal volume of 100 mM sodium citrate pH 5.6, 22%(wt/vol) PEG4000, and 5% (vol/vol) isopropanol. Crystals were harvested inreservoir solution containing 5% (vol/vol) glycerol and flash frozen.

X-ray Crystallographic Analysis and Refinement. Complete datasets werecollected at the SBC-ID19 beamline (Table S1) and processed using HKL3000(51). The crystal structure of hTRAP1N-ADPNP was determined by molecularreplacement using PHENIX (52) and yeast cytosolic Hsp90N (PDB ID code 2CG9)(20) as the search model. The crystal structure of the coiled-coil hTRAP1N dimerwas determined using the structure of hTRAP1N-ADPNP as a search model.Structures were refined using PHENIX (52) and REFMAC5 (53), with 5% of thedata excluded from refinement for cross-validation purposes, which was in-terspersed by several rounds of manual model building in Coot (54). Watermolecules were fitted automatically. The refined structures have excellentstereochemical properties, with none of the residues in generously allowedor disallowed regions of the Ramachandran plot.

In Silico Modeling. To generate amodel of intact hTRAP1, the twofold axis of thecoiled-coil hTRAP1N dimer was aligned with that of intact Hsp90 chaperonestructures to identify a compatible Hsp90MC conformation. The coiled-coil hTRAP1Ndimer was then rotated around the twofold axis until the C termini of hTRAP1Nwere positioned closest to the N termini of Grp94MC avoiding any steric clash.

Analytical Size Exclusion Chromatography. The oligomeric states of hTRAP1Nand hTRAP1NΔ107 (25 mg/mL) were determined at 4 °C in 50 mM Tris·HCl pH 7.5,100mMNaCl, and 1mMDTT on a Superdex 75 10/300 GL column (GE Healthcare).

Disulfide Cross-Linking. hTRAP1, hTRAP1*, or Cys-containing hTRAP1* variantat 0.1 mg/mL was preincubated for 60 min on ice in 40 mM Tris·HCl pH 7.5,

ATP

Pi

ADP

DD T D

T

DD

T T ATP

TT

ATP

ATPPi

Open-state(Ref. 18)

Coiled-coilThis study (Fig. 2A, 3B top)

Closed-state(Ref. 21)

ATP-closed(Ref. 20)

ADP-open(Ref. 33)

ADP-closed(Ref. 18)

ATP-openThis study (Fig. 1A)

Fig. 6. Schematic of the hTRAP1 cycle. TRAP1 subunits are shown in dif-ferent hues and colored red (TRAP1N), green (TRAP1M), and blue (TRAP1C).“T” and “D” indicate bound ATP and ADP, respectively.

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50 mM KCl, 5 mM MgCl2, and 0.2 mM TCEP without or with 5 mM ADPNP,followed by incubation for 10 min at 37 °C. The addition of TCEP was nec-essary to prevent nonspecific cross-linking of Cys-containing hTRAP1* vari-ants. Disulfide bond formation was facilitated by incubating the sample with100 μM copper-o-phenanthroline (Cu:Phe) for 5 min at 23 °C and thenquenching with 20 mM EDTA (final). Cross-linked products were analyzedby nonreducing SDS/PAGE (3–8%) and Coomassie blue staining.

ATPase Assay. ATPase activity was determined at 30 °C by measuring theamount of inorganic phosphate released after 30 min using the malachitegreen calorimetric assay (55) in refolding buffer (30 mM Hepes pH 7.5, 50 mMKCl, 5 mMMgCl2, and 2 mM DTT) containing 5 μM hTRAP1 or hTRAP1 variantsand 2 mM ATP. For mixing experiments, hTRAP1 and hTRAP1 variants weremixed at indicated ratios, keeping the total protein amount constant. Hetero-dimers were incubated for 15 min at 22 °C before the assay.

Chaperone Assay. Chaperone activity was measured by monitoring the re-covery of heat-denatured FFL using a coupled-chaperone assay consisting ofHsp90 chaperones and untreated RRL (Promega), or the bacterial [DnaK/

DnaJ/GrpE (KJE)] or mitochondrial Hsp70 system (Mortalin/Mdj1/Mge1). Inbrief, 167 nM FFL (Promega) was mixed with 20 μM hTRAP1, hHsp90, orbacterial Hsp90 (bHsp90) in the absence or presence of 5 mM ATP in de-naturing buffer (30 mM Tris·HCl pH 7.5 and 2 mM DTT), and heat-denaturedfor 5 min at 45 °C. The samples were cooled on ice for 5 min and then diluted10-fold in refolding buffer containing 50% RRL or 4 μM KJE with 5 mM ATP,or 4 μM Mortalin/Mdj1/Mge1 and an ATP-regenerating system (20 mM cre-atine phosphate, 0.12 mg/mL creatine kinase, and 5 mM ATP). Recovery ofFFL activity was measured at 30 °C every 20 min over 120 min using an LS55fluorescence spectrophotometer (PerkinElmer).

ACKNOWLEDGMENTS. We thank Drs. D. Toft, S. Felts, J. Silberg, J. Barral,and E. Craig for expression constructs. Use of the Structural BiologyCenter (SBC) beamlines at the Advanced Photon Source was supportedby the US Department of Energy, Office of Biological and Environmen-tal Research (Contract DE-AC02-06CH11357). This work was supportedby the National Institutes of Health (Grants R01 GM111084 andR01 GM104980) and the Welch Foundation (Grant Q-1530). J.L. wasa Welch fellow.

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