Mitochondrial Hsp90 Is a Ligand-Activated Molecular Chaperone Coupling ATP Binding to Dimer Closure Through a Coiled-Coil Intermediate

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Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

Nuri 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 review August 14, 2015)

Heat-shock protein of 90 kDa (Hsp90) is an essential molecular major role of TRAP1 in tumorigenesis, although TRAP1’sspecific chaperone that adopts different 3D structures associated with function remains poorly understood (28). distinct nucleotide states: a wide-open, V-shaped dimer in the TRAP1 is a multidomain protein consisting of an N-terminal or N apo state and a twisted, N-terminally closed dimer with ATP. domain (TRAP1N), a middle domain (TRAP1M), and a C-terminal Although the N domain is known to mediate ATP binding, how or C domain (TRAP1C), but it lacks the charged linker found Hsp90 senses the bound nucleotide and facilitates dimer closure in eukaryotic Hsp90 paralogs. Human TRAP1 is preceded by a remains unclear. Here we present atomic structures of human mitochondrial localization sequence (MLS) of 59 residues that are cleaved off during import (29). The mature form of TRAP1 is mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer con- a homodimer held together by TRAP1C, with a second, ATP binding- formation that may precede dimer closure and is conserved in dependent dimer interface in TRAP1N. The crystal structures of intact TRAP1 in solution. Our structure suggests that TRAP1 nor- zebrafish TRAP1 (zTRAP1) and zebrafish and human TRAP1NM

bound to ADPNP were recently reported (21, 30), and are largely BIOPHYSICS AND mally exists in an autoinhibited state with the ATP lid bound to

consistent with our current understanding of Hsp90 chaperones. COMPUTATIONAL BIOLOGY the nucleotide-binding pocket. ATP binding displaces the ATP lid cis In the ATP-bound state, the N-terminal extension (known as the that signals the -bound ATP status to the neighboring subunit N strap) straddles the N domain of the neighboring subunit, in a highly cooperative manner compatible with the coiled-coil thereby stabilizing the structure of the closed-state dimer (21, 30). intermediate state. We propose that TRAP1 is a ligand-activated The ordered segment of the N strap significantly lengthens the molecular chaperone, which couples ATP binding to dramatic previously observed β-strand swap and may function as a regula- changes in local structure required for protein folding. tory element that controls TRAP1 function (16, 21). Interestingly, intact zTRAP1-ADPNP crystallized as an asymmetric dimer that TRAP1 | Hsp90 | molecular chaperone could support a sequential ATP hydrolysis mechanism (31, 32);

eat-shock protein of 90 kDa (Hsp90) is a conserved ATP- Significance Hdependent molecular chaperone (1–4), which together with – heat-shock protein of 70 kDa (Hsp70) (5 7) and a cohort of Mitochondrial heat-shock protein of 90 kDa (Hsp90) (TRAP1) – cochaperones (8 10), promotes the late-stage folding of Hsp90 promotes cell survival and is essential for neoplastic growth. client proteins (11). It is presumed that almost 400 different Exploiting human TRAP1 for drug development requires de- proteins, including a majority of signaling and tumor promoting tailed structural and mechanistic understanding. Whereas TRAP1 proteins, depend on cytosolic Hsp90 for folding (12). Conse- adopts different conformations associated with distinct nucleo- quently, the ability to inactivate multiple oncogenic pathways tide states, how the TRAP1 dimer senses the bound nucleotide simultaneously has made Hsp90 a major target for drug de- and signals this information to the neighboring subunit remains velopment (13), with several Hsp90 inhibitors currently un- unknown. We show that unliganded TRAP1 forms a previously dergoing clinical trials (14). unobserved coiled-coil dimer and is found in an autoinhibited Hsp90 chaperones display conformational plasticity in solution state. ATP binding in cis displaces the ATP lid that signals the (2, 15, 16), with different adenine nucleotides either facilitating nucleotide status to the trans subunit. Our findings suggest that – or stabilizing distinct Hsp90 dimer conformations (17 19). In- human TRAP1 is a ligand-activated molecular chaperone, which terestingly, apo Hsp90 forms a wide-open, V-shaped dimer couples ATP binding to local changes in structure facilitating with the N domains separated by as much as 101 Å (18). This dimer closure needed for protein folding. open conformation is markedly distinct from the intertwined,

N-terminally closed dimer with ATP bound (20, 21). Because Author contributions: N.S., J.L., S.L., and F.T.F.T. designed research; N.S., J.L., J.-H.K., C.C., the open-state dimer cannot signal the nucleotide status between and S.L. performed research; N.S., J.L., J.-H.K., C.C., A.J., S.L., and F.T.F.T. contributed new neighboring subunits, an intermediate conformation preceding reagents/analytic tools; N.S., J.L., C.C., A.J., S.L., and F.T.F.T. analyzed data; and N.S., J.L., dimer closure must exist, which so far has remained elusive. J.-H.K., C.C., A.J., S.L., and F.T.F.T. wrote the paper. Apart from cytosolic Hsp90s, Hsp90 homologs are found in the The authors declare no conflict of interest. endoplasmic reticulum, chloroplasts, and mitochondria (Fig. S1) (22). This article is a PNAS Direct Submission. M.S. is a guest editor invited by the Editorial The tumor necrosis factor receptor-associated protein 1 (TRAP1) is Board. the mitochondrial Hsp90 paralog, which prevents apoptosis and Data deposition: The atomic coordinates and structure factors have been deposited in the protects mitochondria against oxidative damage (23–25). TRAP1 is Protein Data Bank, www.pdb.org (PDB ID codes 5F5R and 5F3K). widely expressed in many tumors (24, 26, 27), but not in mitochon- 1Present address: Pennington Biomedical Research Center, Louisiana State University, dria of most normal tissues (24), benign prostatic hyperplasia (26), or Baton Rouge, LA 70808. highly proliferating, nontransformed cells (27). Notably, it was found 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. that TRAP1 not only promotes neoplastic growth, but also confers This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tumorigenic potential on nontransformed cells (27), indicating a 1073/pnas.1516167113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1516167113 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 however, no asymmetric nucleotide binding was observed, and no the trans segment in the ADPNP-bound TRAP1 dimer (Fig. 1C). molecular contacts between cis-bound ADPNP and the N domain Although this finding is surprising at first glance, the atomic struc- of the neighboring subunit were seen in TRAP1 (21, 30) and other tures of monomeric and dimeric hTRAP1N- and hTRAP1NM- known Hsp90 structures (18, 20, 33), leaving open the question ADPNP complexes are consistent with the notion that hTRAP1- of how TRAP1 senses and signals the nucleotide-bound status ADPNP can adopt both closed- and open-state dimers, with elevated between subunits. temperatures favoring the closed-state conformation (16). In- Here we present atomic structures of human TRAP1N terestingly, a cis-bound N-strap conformation also has been (hTRAP1N) alone and in complex with ADPNP. Unexpectedly, we observed in crystal structures of hTRAP1NM-inhibitor complexes found that unliganded hTRAP1N forms a previously unobserved (30), suggesting that this conformation might be a common feature coiled-coil dimer that is distinct from the proposed open-state and of monomeric TRAP1. closed-state conformations (16, 21, 30). Importantly, intact hTRAP1 Another notable difference is the open conformation of the ATP forms a similar coiled-coil dimer in solution, but only in the absence lid (residues 177 to 202), which exposes the bound nucleotide to of ATP. Our findings show that ATP binding triggers a dramatic bulk solvent (Fig. 1A) as opposed to being folded over the nucle- change in local structure and displaces the ATP lid, which is bound otide-binding pocket (Fig. 1B). A similar open-lid conformation was to the ATP-binding pocket, indicating that TRAP1 normally exists in also reported for monomeric Grp94N-nucleotide complexes (34), an autoinhibited state. Strikingly, mutations of conserved residues suggesting that lid closure is not critical for nucleotide binding to that impair lid binding stimulate the hTRAP1 ATPase activity in a Hsp90 chaperones in general. To determine the structural basis for highly cooperative manner, supporting a previously unknown role of lid closure, we compared the crystal structure of the hTRAP1N- the ATP lid in signaling the cis-bound nucleotide status to the trans ADPNP monomer with that of the zTRAP1-ADPNP dimer (21). subunit, which is compatible with the coiled-coil dimer. Finally, we Superposition of the two structures shows that an open-lid confor- demonstrate that TRAP1 folding requires ATP and the functional mation is incompatible with the closed-state dimer, because it would cooperation of the mitochondrial Hsp70 chaperone system, sup- sterically clash with the N domain of the neighboring subunit (Fig. porting the existence of a mitochondrial Hsp90-Hsp70 supercomplex 1C). Thus, our structure confirms that lid closure is nonessential for that may present a new target for drug development. nucleotide binding and is driven largely by steric interference.

Results Atomic Structure of hTRAP1N Reveals a Coiled-Coil Dimer. In addition Atomic Structure of Monomeric hTRAP1N in the ATP State. The to the ATP-bound state, we report the first, to our knowledge, emerging role of TRAP1 as a potent drug target necessitates atomic structure of unliganded hTRAP1N at 1.82-Å resolution atomic structure information of the human protein to exploit this (Table S1). Unexpectedly, the crystal structure reveals a previously information to develop mitochondrial Hsp90 inhibitors. The 3.3-Å unobserved coiled-coil dimer in the asymmetric unit of the crystal A crystal structure of a closed-state human TRAP1NM (hTRAP1NM) (Fig. 2 ), which is markedly distinct from the proposed open- and dimer bound to ADPNP, which shares close overall similarity with closed-state conformations (16, 21, 30). The coiled-coil dimer in- the previously determined crystal structure of the zTRAP1NM- terface is stabilized by an extensive network of hydrogen bonds ADPNP dimer (21), was reported recently (30). Conversely, formed between the N-terminal helices from each subunit, and 2 2 hTRAP1N-ADPNP crystallized as a monomer (Fig. 1A and occludes 1,765 Å of solvent accessible area. The latter is 38 Å 2 Fig. S2A), with crystals diffracting to 1.85-Å resolution (Table S1). more than the closed-stated zTRAP1N-ADPNP dimer (1,727 Å ) The absence of a dimer is not the result of TRAP1 truncation, as calculated with PISA (35) over the corresponding range of res- however, given that TRAP1N-ADPNP can also crystallize as an idues (zTRAP1N residues 97 to 309), not taking into account an intertwined N-domain dimer (Fig. S3) indistinguishable from that additional 1,218 Å2 owing to contributions of N-strap resi- observed with intact zTRAP1-ADPNP (21). The 2Fo-Fc map of dues (zTRAP1N residues 85 to 96), which are disordered monomeric hTRAP1N is of excellent quality (Fig. S4), enabling in hTRAP1N. tracingofallbutthefirst10residuesofthematureformof In addition to the previously unobserved dimer interface, it is hTRAP1N (Fig. S2A). The sole base-specific interaction between immediately evident that each hTRAP1N monomer adopts a more hTRAP1N andboundADPNPisobservedwiththecarboxylateside- extended, solvent-exposed conformation than previously observed chain of Asp158 that is evolutionary conserved (Fig. S1)andmakes ADPNP-bound complexes (21, 30) (Fig. 1A). Perhaps most a hydrogen bond with the exocyclic N6-amine of adenine (Fig. 1A). strikingly, the N strap no longer forms a β-strand as seen in the α B Pairwise comparison of hTRAP1N and the N domain of intact ADPNP-bound state, but instead forms an -helix (Fig. 2 and zTRAP1 (PDB ID code 4IPE) (21) shows that the two structures Fig. S2B) and pairs up with the helical N strap of the other subunit superpose with an rmsd of only 0.7 Å over 148 Cα atoms. How- to form a zipper-like, coiled-coil dimer (Fig. 2A). The coiled-coil ever, hTRAP1N crystallizes with the N strap bound in cis interface is further stabilized by interactions between Phe183 in (Fig. 1A), as opposed to straddling the N domain in trans (Fig. 1B), the ATP lid of the trans subunit and Phe90, Glu93, and Thr94 of but occupies the same binding site in the hTRAP1N monomer as the cis subunit.

ACB

D158 D173 180o N N N N

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 and zTRAP1-ADPNP showing that an extended ATP lid would sterically clash with the neighboring subunit (gray) in the closed-state dimer.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1516167113 Sung et al. Downloaded by guest on October 1, 2021 A B Lid 3 1 2 90o N N 2 N N N N-terminal N Helix

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

The coiled-coil dimer structure was unexpected, given the com- The N-Terminal α-Helices of Intact hTRAP1 Form a Coiled Coil in the mon presumption that Hsp90N is a monomer in the absence of Absence of Nucleotide. Our attempts to crystallize intact hTRAP1 nucleotide (36), a conformation supported by the apo structure of without nucleotide were unsuccessful. Thus, to generate an in full-length bacterial Hsp90 (18), which shows a wide-open, V-shaped silico model for hTRAP1, we examined previously reported dimer conformation. Consistently, superposing unliganded hTRAP1N structures of full-length Hsp90 chaperones to identify an Hsp90MC onto the equivalent domain of zTRAP1-ADPNP (21) and TRAP1 conformation compatible with the coiled-coil hTRAP1N dimer homologs (18, 20, 33) shows that the helical N strap will sterically structure. Because the structure of the coiled-coil intermediate has clash with the neighboring N domain in the closed-state dimer not been observed previously, it seemed unlikely that we would (Fig. 2B and Fig. S5 A and B), whereas in the proposed open state, find a compatible structure. Fortuitously, several crystal structures the N domains are too far apart to contact each other. were compatible with the imposed twofold restraint. We found To exclude the possibility that the coiled-coil dimer results that the X-ray structures of Grp94 in complex with ADP (PDB ID from crystal packing interactions, we determined the oligo- code 2O1V) or ADPNP (PDB ID code 2O1U) (33) fit best. The BIOPHYSICS AND COMPUTATIONAL BIOLOGY meric state of hTRAP1N in solution. Consistent with the crystal two structures are nearly identical to each other and superpose structure, we found that unliganded hTRAP1N is a dimer, with an rmsd of only 0.5 Å over all Cα atoms. In our model, the C whereas an N-terminally truncated hTRAP1N variant lacking the termini of hTRAP1N (Thr294 and Thr294′) are positioned to their N-terminal α-helix (hTRAP1NΔ107) is monomeric (Fig. 3A). respective N termini of Grp94MC (Leu339 and Leu339′) with an It has been reported that an N-strap truncation (hTRAP1Δ84) equal distance of only 4.3 Å (Fig. 3C, Top and Fig. S6). stimulates ATPase activity by ∼30-fold compared with the mature To validate our model, we wished to probe the coiled-coil form of hTRAP1 (hTRAP1Δ59)(16)(Fig.3B). Strikingly, we found dimer interface of intact hTRAP1 in solution. Because a coiled- that further deletion of the N-terminal α-helix (hTRAP1Δ107)or coil dimer is observed only in the unliganded state, we engineered only the helical N strap (hTRAP1Δ98) nearly abolished the ATPase hTRAP1 variants featuring a Cys in the helical N strap, which activity of hTRAP1 (Fig. 3B). Our findings demonstrate that the potentially could form a disulfide cross-link in the absence of helical N strap has an essential regulatory function and is required nucleotide. In the crystal structure, Lys95 and Leu98 are near the for formation of the coiled-coil dimer that may represent an in- cross-point of the coiled coil with Cα distances of 7.3 Å (Lys95- termediate conformation preceding dimer closure. Lys95′) and 7.5 Å (Leu98-Leu98′), compatible with disulfide

158 44 17 kDa L98 A C S84 2.0 hTRAP1N hTRAP1NΔ107 1.5 S107 1.0 T294 L339 L98C Protein (mg/ml) 0.5 S84 7.5Å 0 K95C o 8 101214161820 90 L98 B Elution Volume (ml) Fig. 3. Human TRAP1N and hTRAP1 form a coiled- 23.6 coil dimer. (A) Size-exclusion chromatogram of 23.4 7.3Å hTRAP1N (black curve) and hTRAP1NΔ107 (gray curve). S107 1.0 (Inset) The hTRAP1N dimer, with the N-terminal 0.8 Δ 0.6 helices deleted in hTRAP1N 107 shown in gray. (B) 0.4 Relative ATPase activities of hTRAP1 (Δ59) and 0.2 0 N-terminal truncated hTRAP1 mutants lacking the

Relative ATPase activity Relative ATPase Δ Δ Δ Δ98 first 84 ( 84), 98 ( 98), or 107 ( 107) residues. (C)In Δ84 Δ107 hTRAP1(Δ59) silico model of the intact coiled-coil hTRAP1 dimer (Top) and the crystal structure of the closed-state D 95C 17.1Å K L98C K95CL98C K95CL98C zTRAP1-ADPNP dimer (21) (Bottom). TRAP1 is shown K95C N RAP1* L98C in blue and magenta; TRAP1MC, in different shades of hTRAP1hTRAP1*hTRAP1*hTRAP1*hTRAP1*hTRAP1*hTRAP1*hTRAP1*hT hTRAP1* gray. Introduced Cys sites are marked by spheres. apo apo ADPNP Cu:Phe + + + + o Corresponding Cys pairs are shown in the same 90 DTT - + - - color, with distances between Cα atoms indicated. kDa 142583 69107 180 (D) Disulfide cross-linking of hTRAP1 and Cys-containing 2mer 135 15.4Å hTRAP1* variants without or with ADPNP. Cross-linked 100 dimers can be monomerized with 100 mM DTT (+DTT). 75 1mer 63 Bands corresponding to hTRAP1 monomers (1mer) and 48 dimers (2mer) are indicated.

Sung et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 N6-amine of adenine in the ADPNP-bound state, and formed a A hydrogen bond with the carboxylate side chain of Asp158 (Fig. 4A).

F201 Because the ATP lid has been implicated in the regulation of Hsp90 ATPase activity (40, 41), we reasoned that the conserved G2 box motif might function as a sensor of nucleotide binding, Q200 G202 D158 providing a means to signal the cis-bound nucleotide status to the neighboring subunit. Consistent with a coordinated ATP hydrolysis mechanism (21, 31), hTRAP1 variants impaired in lid BDC 1.0 binding should stimulate ATPase activity by signaling a consti- 0.6 4.0 cis trans 0.8 tutive -bound nucleotide status to the subunit. 3.0 0.4 0.6 To test this idea, we mutated Asp158, Gln200, and Phe201 either 2.0 0.2 0.4 alone or in combination. We found that our engineered hTRAP1 1.0 D158A B C ATPase rate ATPase ATPase rate ATPase 0.2 D158A/F201A variants retained ATPase activity (Fig. 4 and ), including an 0 0

ATPase activity ATPase Theoretical 0 hTRAP1 variant featuring a mutation of Asp158 to Asn. Although 6:0 5:1 4:2 3:3 2:4 1:5 0:6 in principle, an Asn is compatible with ATP binding, our finding Q200AF201A D158ND158WD158YD158FD158Q D158A Wild type Wild type [hTRAP1]:[hTRAP1 variant] F201A differs from previous observations with cytosolic Hsp90 (42, 43). Q200A/F201A D158A/Q200AD158A/F201A D158A/Q200A/ Surprisingly, hTRAP1Q200A/F201A showed only 4.1-fold higher ATPase activity than hTRAP1, slightly less than that observed with Fig. 4. Conserved residues of the ATP-lid G2 box motif compete with ATP hTRAP1 alone (Fig. 4B), indicating that lid binding is driven for binding to the ATP-binding pocket. (A) Superposition of unliganded and F201A largely by van der Waals interactions. Interestingly, we found that the ADPNP-bound hTRAP1N. Only ADPNP of the hTRAP1N-ADPNP complex is shown. (B and C) ATPase rates, expressed in μmol ATP hydrolyzed per min ATPase activity of TRAP1D158N was increased only slightly (2.5-fold), μ whereas a mutation of Asp158 to Ala stimulated ATPase activity by per mol protein of hTRAP1 and hTRAP1 variants carrying a mutation in C Gln200 or Phe201 (B) and/or Asp158 (C). Averages of three independent 14.6-fold (Fig. 4 ), suggesting that the stimulated ATPase activity of measurements ± SD are shown. (D) Relative ATPase activities of mixtures of hTRAP1 variants depends on the nature of the introduced substitution.

hTRAP1 and hTRAP1D158A (red), and hTRAP1 and hTRAP1D158A/F201A heter- The greatest stimulation was observed with hTRAP1D158A/F201A, odimers (cyan) at indicated ratios. The dashed line represents the theoreti- which increased ATPase activity by 33.0-fold, significantly greater cal, linear relationship if the stimulated ATPase activities resulted from an than the increases seen with hTRAP1D158A/Q200A/F201A triple increase in ATPase activity in only one subunit. mutant (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, C Top bond formation (Fig. 3 , ), as opposed to 17.1 Å and 15.4 Å we generated heterodimers of hTRAP1 and hTRAP1D158A and of C Bottom in the ADPNP-bound state (21) (Fig. 3 , ). Thus, we hTRAP1 and hTRAP1D158A/F201A by mixing wild-type and mutant generated hTRAP1 mutants by replacing Lys95 or Leu98 with subunits in defined molar ratios, keeping the total protein amount Cys in a Cys-free hTRAP1 variant (hTRAP1*), which is fully constant. We would expect to observe a linear relationship if the functional (Fig. S7). Notably, hTRAP1*K95C and hTRAP1*L98C stimulated ATPase activity resulted from an increase in activity formed a cross-linked dimer only in the absence of nucleotide within one subunit, and a nonlinear relationship if stimulation (Fig. 3D, lanes 6 and 7), but not in the presence of ADPNP (Fig. occurred between neighboring subunits. As shown in Fig. 4D,we 3D, lanes 9 and 10). The cross-linking reaction is reversible with observed a clear nonlinear relationship with both the hTRAP1/ DTT (Fig. 3D, compare lanes 6 and 7 with lanes 3 and 4), and no hTRAP1D158A and hTRAP1/hTRAP1D158A/F201A heterodimers. cis cross-linked products were observed with hTRAP1 (Fig. 3D, lane Thus, mutations that impair lid binding in stimulate the trans 1), underscoring the specificity of the cross-linking reaction. ATPase activity in . Taken together, our findings suggest that the ATP lid competes with nucleotide binding and is displaced by Taken together, our findings lead us to conclude that unliganded cis hTRAP1 adopts a similar coiled-coil dimer in solution as ob- ATP, providing a means to signal the -bound nucleotide status served with hTRAP1 in the crystal. to the neighboring subunit that must be in close physical prox- N imity, a conformation that is compatible with the coiled-coil dimer. The ATP Lid Functions as a Key Regulatory Element. The crystal structure of TRAP1 confirmed that the nucleotide-binding do- TRAP1 Cooperates with Mitochondrial Chaperones in Folding. To determine the significance of the unliganded and ATP-bound main shares the canonical Bergerat fold (37), which features TRAP1 conformation, we developed an assay to monitor the ATP- several conserved motifs, including the G1 box and G2 box that dependent hTRAP1 chaperone activity. It is known that cytosolic flank the ATP lid (Fig. S1). It was initially proposed that the lid Hsp90 requires Hsp70 and a cohort of cochaperones to promote represents an ATP-binding motif (37) that folds over the nucleotide-binding pocket to stabilize bound ATP in GHKL (gyrase, Hsp90, histidine kinase, MutL) ATPases (38, 39). Although deletion of the lid did not affect nucleotide binding to Hsp90, + RRL + Mortalin/ AB Mdj1/Mge1 lidless yeast Hsp90 lacks ATPase activity and cannot rescue an 30 + KJE 50 TRAP1-ATP − − hsp82 /hsc82 40 yeast strain (40), underscoring the importance of 20 the ATP lid to Hsp90 function. 30 10 In the crystal structure of hTRAP1N-ADPNP, the ATP lid is 20

folded away from the nucleotide-binding pocket, consistent with the % Reactivated FFL 0 TRAP1 10

notion that the lid is dispensable for ATP binding. Interestingly, in % Reactivated FFL 0 no TRAP1 the crystal structure of unliganded hTRAP1N,theATPlidfolds 0 20 40 60 80 100 120 hHsp90/apohHsp90/ATPhTRAP1/apobHsp90/apobHsp90/ATPbHsp90/ATP over the nucleotide-binding pocket. Closer inspection reveals that no protein/apo hTRAP1/ATP Time (min) Gln200, Phe201, and Gly202 of the G2 box overlapped with the α A Fig. 5. TRAP1 requires the mitochondrial Hsp70 system for protein folding. ribose ring and the -phosphate of the bound nucleotide (Fig. 4 ), ± effectively competing with nucleotide binding (Fig. S8A). A similar Averages of three independent measurements SD are shown. (A) Reac- tivation of heat-denatured FFL after 120 min by hHsp90, hTRAP1, or bHsp90 G2 box motif interaction was also observed in the crystal structure B together with RRL or the bacterial Hsp70 system (KJE). Recovered FFL ac- of full-length bacterial Hsp90 in the apo conformation (Fig. S8 ) tivities are expressed relative to native FFL. (B) Reactivation of heat-dena- (18), indicating that mitochondrial and bacterial Hsp90 may nor- tured FFL without hTRAP1 (no TRAP1), with hTRAP1 (TRAP1), or with mally exist in an autoinhibited state. Interestingly, in hTRAP1, the hTRAP1 and 5 mM ATP (TRAP1-ATP), and together with Mortalin/Mdj1/ amine group of the Gln200 side chain superposed with the exocyclic Mge1. FFL recovery was monitored every 20 min for 120 min.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1516167113 Sung et al. Downloaded by guest on October 1, 2021 ATP-open Open-state Coiled-coil the Gln200 side chain mimics the bound nucleotide and con- This study (Fig. 1A) (Ref. 18) This study (Fig. 2A, 3B top) tributes toward lid binding by forming a hydrogen bond with the Asp158 side chain (Fig. 4A). Although an inhibitory lid confor- ATP ATP T TT mation also has been observed with full-length bacterial Hsp90 (18), the interaction between the conserved Gln200 and Asp158 side chains is new. Consistent with a nucleotide sensor function, Pi ADP ATP ATP hTRAP1 variants carrying a mutation in the conserved Asp158 or Phe201, which impairs lid-binding, show highly stimulated B C D D D D T D T T ATPase activity (Fig. 4 and ). On the other hand, substituting Gln200 with Ala has a negligible effect on ATPase activity, and hTRAP1 variants carrying an additional mutation in Asp158, Pi Phe201, or both exhibit similar or reduced ATPase activity com- ADP-closed ADP-open Closed-state ATP-closed pared with those that do not feature the Gln200 mutation (Fig. 4 B (Ref. 18) (Ref. 33) (Ref. 21) (Ref. 20) and C). Taken together, these findings point to an additional role Fig. 6. Schematic of the hTRAP1 cycle. TRAP1 subunits are shown in dif- for Gln200 in the ATPase cycle. In the crystal structure of zTRAP1-ADPNP (PDB ID code 4IPE-A) (21), the equivalent ferent hues and colored red (TRAP1N), green (TRAP1M), and blue (TRAP1C). “T” and “D” indicate bound ATP and ADP, respectively. glutamine forms water-mediated interactions with the main chain of a β-bridge featuring the ATP sensor (Arg402) (48). Thus, we speculate that, in addition to lid binding, Gln200 is needed to the folding of Hsp90 clients (1, 10). Interestingly, hTRAP1 cannot position the ATP-sensor loop required for full ATPase activity. functionally replace cytosolic Hsp90 in vitro (29, 44), suggesting the Finally, we have demonstrated that TRAP1-dependent protein need for other factors to reconstitute TRAP1-dependent folding. folding requires ATP (Fig. 5B). ATP hydrolysis may be needed for Rabbit reticulocyte lysate (RRL) provides a rich source of pro- substrate release, as has been reported for cytosolic Hsp90 (49). In teins, and can substitute for Hsp70 and essential cochaperones to addition to ATP, TRAP1-dependent protein folding also requires reconstitute active folding by cytosolic Hsp90 in vitro (45). Although the functional cooperation of the mitochondrial Hsp70 system. RRL facilitated hHsp90-dependent folding of heat-denatured The latter supports a direct physical interaction, as was recently firefly luciferase (FFL), no recovered FFL activity was observed reported for cytosolic Hsp90 chaperones (5–7, 50), and opens up when hHsp90 was replaced with either hTRAP1 or bacterial Hsp90 new avenues for drug development by targeting the specific in-

(Fig. 5A). However, bacterial Hsp90 could recover FFL activity teraction between mitochondrial chaperones. BIOPHYSICS AND A

when RRL was replaced with the bacterial Hsp70 system (Fig. 5 ). COMPUTATIONAL BIOLOGY Although perplexing on first sight, it has been reported that Experimental Procedures RRLs mostly lack functional mitochondria (46). Thus, we rea- Cloning, mutagenesis, and protein expression and purification procedures soned that our inability to reconstitute TRAP1-dependent protein are described in SI Experimental Procedures. folding with RRL might be due to the lack of one or more com- ponents of the mitochondrial chaperone system. Indeed, we found Crystallization. Human TRAP1N crystals were grown at 14 °C from hanging μ · that hTRAP1 reactivated heat-denatured FFL when RRL was drops. Then 2 L of hTRAP1N (25 mg/mL) in 30 mM Tris HCl pH 8.5, 0.15 M replaced with the mitochondrial Hsp70 system (Fig. 5B). Impor- NaCl, and 1 mM tris(2-carboxyethyl)phosphine (TCEP) was mixed with an tantly, hTRAP1’s ability to reactivate FFL requires ATP during equal volume of reservoir solution consisting of 100 mM Hepes pH 7.5, μ heat denaturation, whereas the presence of unliganded hTRAP1 100 mM CaCl2, 23% (wt/vol) PEG 3350, 4% (vol/vol) isopropanol, and 0.4 L and the absence of hTRAP1 resulted in very low and no recovered of 1 M LiCl. hTRAP1N crystals were harvested in reservoir solution containing FFL activity, respectively (Fig. 5B). 5% (vol/vol) PEG400, but reducing the PEG 3350 concentration to 21% (wt/vol), and flash frozen in liquid nitrogen. hTRAP1N-ADPNP was prepared · Discussion by incubating hTRAP1N (55 mg/mL) in 50 mM Tris HCl pH 7.6, 0.2M NaCl, and 1 mM TCEP with 5 mM ADPNP and 10 mM MgCl2 for 30 min on ice. The sample Our findings provide mechanistic insight into how ATP-binding– was mixed with an equal volume of 100 mM sodium citrate pH 5.6, 22% induced local changes in TRAP1 structure facilitate dimer closure, (wt/vol) PEG4000, and 5% (vol/vol) isopropanol. Crystals were harvested in and suggest that TRAP1 is a ligand (ATP)-activated molecular reservoir solution containing 5% (vol/vol) glycerol and flash frozen. chaperone. Although our results reconcile disparate structural and biochemical observations, they also reveal nuances specific to mi- X-ray Crystallographic Analysis and Refinement. Complete datasets were tochondrial Hsp90. For instance, we find that an hTRAP1 variant collected at the SBC-ID19 beamline (Table S1) and processed using HKL3000 featuring an Asp158-to-Asn mutation behaves differently from that (51). The crystal structure of hTRAP1N-ADPNP was determined by molecular reported for cytosolic Hsp90s (42, 43). Although an Asn in place of replacement using PHENIX (52) and yeast cytosolic Hsp90N (PDB ID code 2CG9) an Asp is not unprecedented among GHKL ATPases (47), it in- (20) as the search model. The crystal structure of the coiled-coil hTRAP1N dimer dicates that the hTRAP1 ATPase activity is fine-tuned differently was determined using the structure of hTRAP1N-ADPNP as a search model. from other Hsp90s. Moreover, the inability to functionally interact Structures were refined using PHENIX (52) and REFMAC5 (53), with 5% of the with known Hsp90 cochaperones suggests that TRAP1 is de- data excluded from refinement for cross-validation purposes, which was in- pendent on other regulatory elements integral to TRAP1, such as terspersed by several rounds of manual model building in Coot (54). Water the N strap (16) and the ATP lid, whose function is uncovered here. molecules were fitted automatically. The refined structures have excellent stereochemical properties, with none of the residues in generously allowed Our findings suggest that TRAP1-ATP can in principle adopt or disallowed regions of the Ramachandran plot. two distinct conformations: a closed-state dimer (ATP closed) with the N strap swapped between subunits (21, 30) and an open- In Silico Modeling. To generate a model of intact hTRAP1, the twofold axis of the state dimer (ATP open) with the N strap bound in cis (Fig. 6), coiled-coil hTRAP1N dimer was aligned with that of intact Hsp90 chaperone which is consistent with the observed conformational plasticity of structures to identify a compatible Hsp90 conformation. The coiled-coil hTRAP1 Hsp90 in solution (2, 15, 16). However, we propose that the ATP MC N dimer was then rotated around the twofold axis until the C termini of hTRAP1N open state does not promote protein folding. were positioned closest to the N termini of Grp94MC avoiding any steric clash. More importantly, our findings support a role of the ATP lid as a sensor of nucleotide binding. ATP binding in cis displaces the ATP Analytical Size Exclusion Chromatography. The oligomeric states of hTRAP1 trans N lid that in turn signals the nucleotide status to the subunit. To and hTRAP1NΔ107 (25 mg/mL) were determined at 4 °C in 50 mM Tris·HCl pH 7.5, do so, the N domains must be in close physical proximity, a 100 mM NaCl, and 1 mM DTT on a Superdex 75 10/300 GL column (GE Healthcare). conformation that is fully compatible with our coiled-coil intermediate dimer. The role of the Gln200 side chain is perhaps Disulfide Cross-Linking. hTRAP1, hTRAP1*, or Cys-containing hTRAP1* variant most intriguing. In the crystal structure of unliganded hTRAP1N, at 0.1 mg/mL was preincubated for 60 min on ice in 40 mM Tris·HCl pH 7.5,

Sung et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 50 mM KCl, 5 mM MgCl2, and 0.2 mM TCEP without or with 5 mM ADPNP, DnaJ/GrpE (KJE)] or mitochondrial Hsp70 system (Mortalin/Mdj1/Mge1). In followed by incubation for 10 min at 37 °C. The addition of TCEP was nec- brief, 167 nM FFL (Promega) was mixed with 20 μM hTRAP1, hHsp90, or essary to prevent nonspecific cross-linking of Cys-containing hTRAP1* vari- bacterial Hsp90 (bHsp90) in the absence or presence of 5 mM ATP in de- ants. Disulfide bond formation was facilitated by incubating the sample with naturing buffer (30 mM Tris·HCl pH 7.5 and 2 mM DTT), and heat-denatured 100 μM copper-o-phenanthroline (Cu:Phe) for 5 min at 23 °C and then for 5 min at 45 °C. The samples were cooled on ice for 5 min and then diluted quenching with 20 mM EDTA (final). Cross-linked products were analyzed 10-fold in refolding buffer containing 50% RRL or 4 μM KJE with 5 mM ATP, by nonreducing SDS/PAGE (3–8%) and Coomassie blue staining. or 4 μM Mortalin/Mdj1/Mge1 and an ATP-regenerating system (20 mM creatine phosphate, 0.12 mg/mL creatine kinase, and 5 mM ATP). Recovery of ATPase Assay. ATPase activity was determined at 30 °C by measuring the amount of inorganic phosphate released after 30 min using the malachite FFL activity was measured at 30 °C every 20 min over 120 min using an LS55 green calorimetric assay (55) in refolding buffer (30 mM Hepes pH 7.5, 50 mM fluorescence spectrophotometer (PerkinElmer).

KCl, 5 mM MgCl2, and 2 mM DTT) containing 5 μM hTRAP1 or hTRAP1 variants and 2 mM ATP. For mixing experiments, hTRAP1 and hTRAP1 variants were ACKNOWLEDGMENTS. We thank Drs. D. Toft, S. Felts, J. Silberg, J. Barral, mixed at indicated ratios, keeping the total protein amount constant. Hetero- and E. Craig for expression constructs. Use of the Structural Biology dimers were incubated for 15 min at 22 °C before the assay. Center (SBC) beamlines at the Advanced Photon Source was supported by the US Department of Energy, Office of Biological and Environmen- tal Research (Contract DE-AC02-06CH11357). This work was supported Chaperone Assay. Chaperone activity was measured by monitoring the re- by the National Institutes of Health (Grants R01 GM111084 and covery of heat-denatured FFL using a coupled-chaperone assay consisting of R01 GM104980) and the Welch Foundation (Grant Q-1530). J.L. was Hsp90 chaperones and untreated RRL (Promega), or the bacterial [DnaK/ aWelchfellow.

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