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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 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.
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