Molecular Cell

Review Hsp90: Breaking the Symmetry Matthias P. Mayer1,* and Laura Le Breton1 1Zentrum

fu¨r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.02.022

Hsp90 chaperones receive much attention due to their role in cancer and other pathological conditions, and a tremendous effort of many laboratories has contributed in the past decades to considerable progress in the understanding of their functions. Hsp90 chaperones exist as dimers and, with the help of cochaperones, promote the folding of numerous client proteins. Although the original view of these interactions suggested that these dimeric complexes were symmetrical, it is now clear that many features are asymmetrical. In this review we discuss several recent advances that highlight how asymmetric interactions with cochaperones as well as asymmetric posttranslational modifications provide mechanisms to regulate client interactions and the progression through Hsp90’s chaperone cycle. Introduction The 90 kDa heat shock protein (Hsp90) family consists of ubiquitous and very abundant ATP-dependent molecular chaperones that are essential for eukaryotic cells. Different isoforms are expressed in different cellular compartments: Hsp90a and Hsp90b in the cytosol and nucleus, Grp94 in the ER, TRAP1 in mitochondria, and Hsp90C in plastids (Johnson, 2012; Stankiewicz and Mayer, 2012; Taipale et al., 2010). Several of the Hsp90 isoforms are induced by the heat shock response or compartment-specific stress response signaling circuits (Nollen and Morimoto, 2002). All isoforms and homologs share a high degree of sequence identity (Johnson, 2012). As many other chaperones, Hsp90 can interact with misfolded proteins and prevent their aggregation. However, this is not their main and essential function and, in contrast to Hsp60s and Hsp70s, they cannot refold misfolded proteins to the native state. Hsp90s, assisted in the eukaryotic cytosol by a large number of cochaperones, act on native-like protein substrates called clients (Johnson, 2012). The current list of clients (http://www. picard.ch/downloads/Hsp90interactors.pdf) includes receptors, transcription factors, kinases, and other unrelated proteins, sharing no common features in terms of sequence or structure (for a complete list of interactors, see http://www.picard.ch/ Hsp90Int/index.php; Echeverrı´a et al., 2011). Hsp90 chaperones determine stability and activity of their clients, many of which take part in signaling pathways and regulatory circuits, controlling cell homeostasis, growth, proliferation, differentiation, and cell death. Among Hsp90 clients are numerous oncoproteins, tumor suppressors, proteins linked to tumor progression, invasiveness, and metastatic potential. Therefore, Hsp90 appears as a promising target in the development of new anti-cancer therapies, and specific inhibitors are in clinical trials (Trepel et al., 2010). Hsp90 is a dynamic dimer (Figure 1), and there is ample evidence that dimerization is essential for its chaperoning function in vivo (Wayne and Bolon, 2007). The Hsp90 protomer consists of three functionally distinct domains. The N-terminal domain (NTD) is involved in nucleotide binding and hydrolysis. Many small-molecule inhibitors of Hsp90’s activity compete 8 Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc.

with ATP for binding to the nucleotide-binding pocket. The NTD is linked to the middle domain (MD) via a disordered region, containing basic and acidic residues, referred to as charged linker (CL). Interestingly, its length is highly variable within the Hsp90 family, ranging from 10 residues in Escherichia coli HtpG to 56 in yeast, 63 in human, and 95 in Plasmodium falciparum Hsp90. Most of its length seems to be dispensable in yeast (Hainzl et al., 2009), and the sequence is not conserved and can be replaced by alternating glycine and serine residues (Tsutsumi et al., 2012). The function of this flexible linker is so far unknown. The MD also plays a concerted role in ATP hydrolysis together with the NTD. The C-terminal domain (CTD) is responsible for constitutive dimerization. All three domains provide interfaces for binding cochaperones and clients. A general model for client interaction with Hsp90 is shown in Figure 2. Clients first interact with Hsp70 in an Hsp40-mediated ATP-hydrolysis-dependent process. Hsp70 transfers the client onto Hsp90 via an intermediate Hsp70-client-Hsp90 complex. Dissociation of Hsp70 leads to the mature Hsp90-client complex, which keeps the client in an inactive but readily activatable state. The Hsp90-client complex may dissociate upon activation of the client or spontaneously, and the free client may reenter the cycle through Hsp70. This chaperone cycle is regulated and modified by a large number of cochaperones, some of which are shown in Figure 2, by exerting different functions. They stimulate or inhibit Hsp90’s ATPase activity, act during client loading as adaptors or scaffolds, and they stabilize the Hsp90-client complex during different stages of the cycle or drive the progression through the chaperone cycle (Table 1). The Hsp90 cycle is fine-tuned by a large number of posttranslational modifications on Hsp90 and cochaperones. Increasing evidence of simultaneous interaction with several cochaperones and independent activity of Hsp90’s protomers made asymmetry emerge as a key in understanding the molecular mechanism of Hsp90 chaperones. In this review, we focus on this asymmetry in Hsp90 complexes, the regulation of Hsp90 by cochaperones and posttranslational modifications, as well as the latest findings on interactions with clients.

Molecular Cell

Review 2CG9

Figure 1. Hsp90 Structure and Conformational Dynamics

NTD

From top to bottom, cartoon representations of yeast Hsp90 in closed conformation (PDB ID: 2CG9; Ali et al., 2006), E. coli Hsp90 in ADP bound state (PDB ID: 2IOP; Shiau et al., 2006), canine Grp94 (PDB ID: 2O1U; Dollins et al., 2007), E. coli Hsp90 in nucleotide-free state (PDB ID: 2IOQ; Shiau et al., 2006), E. coli Hsp90 in wide open conformation according to SAXS data (courtesy of D. Agard; Krukenberg et al., 2008). NTD, N-terminal domain (dark blue and dark teal); CL, charged linker (gray, dashed lines represent parts not present in the structure); MD, middle domain (cyan and greencyan); CTD, C-terminal domain (blue and dark green).

ATP CL

MD

CTD ADP 2IOP

2O1U

2IOQ

2IOQ-SAXS

ADP

Asymmetry in the Hsp90 Conformational Cycle Hsp90 Conformational Dynamics Structural studies revealed that the Hsp90 dimer is highly dynamic and populates distinct conformations, ranging from a wide-open V-shape with a high degree of rotational freedom between NTD and MD and MD and CTD, to a twisted intertwined compact conformation with intimate contacts between both NTDs of the Hsp90 dimer, including exchange of the N-terminal strand, and between the NTD and the MD within each protomer (Figure 1) (Ali et al., 2006; Dollins et al., 2007; Krukenberg et al., 2008; Shiau et al., 2006). The compact state seems to be the ATP hydrolysis-competent conformation in which the nucleotide binding pocket of the NTD is complemented by an arginine from the MD, which contacts the g-phosphate of ATP, stabilizing the catalytic active conformation of the Hsp90 dimer and possibly the transition state of catalysis (Cunningham et al., 2012; Prodromou, 2012). Consistently, amino acid replacements, which stabilize the N-terminally dimerized state, increase the ATPase activity of Hsp90; monomerization by deletion of the CTD or amino acid replacements that disfavor N-terminal dimerization decrease the ATPase activity (Cunningham et al., 2008; Richter et al., 2001; Vaughan et al., 2009). Hsp90 dimers are also able to dissociate in their C-terminal dimer interface, which seems to be influenced by nucleotides and anti-correlated to N-terminal dimerization, indicating long-range intra- and interprotomer communication (Ratzke et al., 2010). How these different conformations are linked into an ATPase cycle has changed in the course of evolution. E. coli HtpG is highly dynamic in the apo state and responds to ATP binding with transition to a tensed state by a ratchet-like mechanism. This indicates a rather tight mechano-chemical coupling between bound nucleotide and conformation, which is characteristic for a deterministic protein machine (Graf et al., 2009; Ratzke et al., 2012). Yeast and human Hsp90s are even more dynamic in the apo state than E. coli HtpG, and nucleotide binding does not lead to a coordinated transition to a tensed state. Instead, independent of the nucleotide bound to the NTD, yeast Hsp90 interconverts between two open and two closed conformations by thermal fluctuations, and nucleotides only slightly change the energy barriers between the different conformations (Graf et al., 2014; Mickler et al., 2009). Therefore, eukaryotic Hsp90s behave like probabilistic protein machines in the absence of regulatory cochaperones. From Symmetry to Asymmetry Homooligomeric protein assemblies are expected to be symmetric (Goodsell and Olson, 2000; Swapna et al., 2012), and the crystal structures of bacterial, yeast, and mammalian fulllength Hsp90 proteins in apo, ADP, and AMPPNP bound states Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc. 9

Molecular Cell

Review p4 Hs

Hop

ATP

ATP

70 ATP

ATP

C

H sp 70

N

sp

0

H

Figure 2. Hsp90 Chaperone Cycle

ATP

Hop

C

? Hs

p4

0

MEEVD

ADP

Cpr6

PPI ase

MEEVD

Hop

C

M

C

Cpr6

p2

3 ligand

ATP

ATP ATP

ATP

sp

ATP

H

p23

ADP

70

PPI

Cpr6

PPI

C

a1

ase

a1 Hop

Ah

were all symmetric, though they were in different conformations as described above (Ali et al., 2006; Dollins et al., 2007; Shiau et al., 2006). The recently solved structure of the mitochondrial Hsp90 isoform TRAP1 from Danio rerio in complex with ATP analogs is the first structure of an isolated Hsp90 protein to exhibit significant asymmetry in the closed, N-terminally dimerized state (Figure 3 and Movie S1) (Lavery et al., 2014). One of the two protomers of TRAP1 (chain B) has a conformation very similar to the previously solved structure of yeast Hsp90, likewise in the closed conformation, while the other protomer (chain A) exhibits significant deviations from symmetry, especially at the NTD-MD and the MD-CTD interfaces. This asymmetry also exists in solution, and replacement of residues involved in asymmetry reduces the ATPase activity of D. rerio and human TRAP1. Moreover, this asymmetry does not seem to be a unique feature of the mitochondrial Hsp90 because SAXS data for E. coli Hsp90 also give indications for existing asymmetry. Therefore, asymmetric states may be a more general feature within the family of Hsp90 proteins and may not have been observed previously because the only other Hsp90 crystallized in the closed state was in complex with two molecules of the cochaperone p23/Sba1 (Ali et al., 2006), which bridge both NTDs of the Hsp90 dimer and influence the conformation of MD and CTD, as observed by NMR and hydrogen exchange mass spectrometry (Graf et al., 2014; Karago¨z et al., 2011). The asymmetry in TRAP1 is linked to binding of ATP and dimerization of NTDs and CTDs because removal of the CTDs leads to a symmetric structure, suggesting that simultaneous N- and C-terminal dimerization induces a strain on the Hsp90 dimer that is relieved by asymmetry in the MD. Agard and col10 Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc.

a1

PPI

Ah

Hs

p7

0

p23

Hop

Ah

Cpr6

Cpr6

ase

C

ase

C

In the absence of cochaperones and clients, Hsp90 transits frequently between open and closed conformations through thermal fluctuations (N, N-terminal domain; M, middle domain; C, C-terminal domain). Binding of a single Hop/Sti1 with its TPR2a domain to the C-terminal MEEVD motif and with its TPR2b domain to the MD of one protomer arrests the Hsp90 dimer in a V-shaped open conformation, preventing N-terminal dimerization and ATP hydrolysis. Hsp70 with a bound client (C) interacts with its C-terminal IEEVD motif with the TPR1 domain of Hop leading to the positioning of Hsp70 between the two NTDs of the Hsp90 dimer and to client transfer. Binding of a PPIase (e.g., Cpr6 or FKBP52) to the second MEEVD motif and presumably MD and maybe NTD primes the Hsp90 dimer for Aha1-induced transition to the closed conformation and rapid dissociation of HOP/Sti1 and Hsp70. A second PPIase (e.g., Cpr6, Cpr7, FKBP51, FKBP52, Cyp40) may bind to the free MEEVD motif and other regions of Hsp90. p23/Sba1 displaces Aha1 and stabilizes the Hsp90-client complex with Hsp90 in the closed intertwined conformation. Ligand binding to the client or other forms of client activation may occur in this complex. ATP hydrolysis, maybe in a sequential fashion in both Hsp90 protomers, leads to cochaperone and client dissociation.

leagues propose that the asymmetry prevents simultaneous ATP hydrolysis in both subunits and imposes a sequential order of hydrolysis (Lavery et al., 2014). This model is consistent with the observation by Mishra and Bolon that survival of yeast depends on ATP binding in both protomers of the Hsp90 dimer, but only one of the two subunits needs to be competent for ATP hydrolysis (Mishra and Bolon, 2014). Interestingly, the MD-CTD interface, which is most affected by the asymmetry, comprises residues that are posttranslationally modified and influence Hsp90 activity, as will be discussed later. However, the observed asymmetry in the isolated TRAP1 could also be restricted to the prokaryotic-type Hsp90s, which have a very short linker between NTD and MD (%10 residues) as compared to eukaryotic Hsp90s (50–90 residues). In addition, eukaryotic Hsp90s are much more conformationally dynamic and do not respond to ATP binding with stringent conformational changes as mentioned above (Graf et al., 2014; Mickler et al., 2009; Ratzke et al., 2012). Any strain induced by simultaneous N- and C-terminal dimerization could also be the driving force for the dynamic opening and closing of the C-terminal dimerization interface, which was not observed for E. coli Hsp90 (Ratzke et al., 2010; 2012). At this stage, it is not yet clear whether asymmetry might be an intrinsic feature of eukaryotic Hsp90s, and addressing this point could prove technically challenging. Alternatively, asymmetry of eukaryotic Hsp90s might only be imposed by interactions with cochaperones, which seem to be absent in bacteria, mitochondria, and plastids. Asymmetry within Hsp90-Cochaperone Complexes The activity of eukaryotic cytosolic Hsp90s is regulated by a large number of cochaperones, some of which act as general

Molecular Cell

Review Table 1. Non-Exhaustive List of Hsp90 Cochaperones Cited in This Review Cochaperone

Binding Site in Hsp90

Function

References

Hop/Sti1

MEEVD motif, CTD, MD, NTD

general cochaperone, inhibits ATPase activity, interaction with Hsp70

Lee et al., 2012; Li et al., 2011; Prodromou et al., 1999; Schmid et al., 2012

FKBP51, FKBP52

MEEVD motif

peptidyl prolyl isomerase, involved in SHR activation

Pirkl and Buchner, 2001

Cpr6, Cyp40/Cpr7

MEEVD motif

peptidyl prolyl isomerase, involved in SHR activation

Mayr et al., 2000; Pirkl and Buchner, 2001

PP5/Ppt1

MEEVD motif

phosphatase, involved in SHR and kinase activation

Soroka et al., 2012; Wandinger et al., 2006

CHIP

MEEVD motif

general cochaperone, ubiquitin ligase, interaction with Hsp70

Min et al., 2008

Aha1

NTD, MD

general cochaperone, stimulates ATPase activity

Li et al., 2013; Meyer et al., 2004; Retzlaff et al., 2010

p23/Sba1

NTD, MD

general cochaperone, inhibits ATPase activity

Echtenkamp et al., 2011; Grad et al., 2006

Cdc37

NTD

involved in kinase activation, inhibits ATPase activity

Siligardi et al., 2002

Sgt1

NTD

adaptor for clients

Zhang et al., 2008

Tah1, Spagh

MEEVD motif

through Pih1, R2TP adaptor for TTT complex

Pal et al., 2014; Zhao et al., 2005

cofactors, while others are dedicated to a subset of clients. The primary interaction site for the largest group of cochaperones, the tetratricopeptide repeat (TPR) domain cochaperones, is the MEEVD motif at the very C terminus of Hsp90, at the end of a long flexible linker, but they also interact with the core of the CTD, with the MD, and potentially with the NTD (Lee et al., 2012; Scheufler et al., 2000; Schmid et al., 2012). Other cochaperones interact primarily with the NTD and/or the MD (Figure 4, Table 1) (Ro¨hl et al., 2013). Most of the cochaperones bind, and thereby stabilize, defined conformations of Hsp90. Thus, they convert the probabilistic conformational fluctuations of Hsp90 into a directional chaperoning cycle. Although many of the Hsp90-cochaperone complexes were considered symmetric originally (e.g., Prodromou et al., 1999), there is now evidence that many cochaperones exert their effect on Hsp90 already in a 1:2 stoichiometry. For example, the TPR domain protein Hop/Sti1 forms 2:2 and 1:2 complexes with Hsp90 (Ebong et al., 2011) and inhibits the ATPase activity of Hsp90 already when a single Hop/Sti1 is bound (Schmid et al., 2012). Interestingly, negative-stain EM images of 2:2 and 1:2 complexes look almost identical in respect to the density for Hsp90, which assumes an open V-shaped conformation. This indicates that a single Hop/Sti1 molecule is sufficient to stabilize Hsp90 in a defined conformation, which is incompetent for ATP hydrolysis, explaining the inhibitory effect of Sti1 on Hsp90’s ATPase activity (Lee et al., 2012; Schmid et al., 2012; Southworth and Agard, 2011). Similarly, Aha1, which interacts with the MD and NTD, stimulates the ATPase activity of both Hsp90 protomers when bound only to a single protomer, presumably by stabilizing the N-terminally dimerized conformation of Hsp90 (Retzlaff et al., 2010). When two different TPR domain proteins are added to Hsp90, heterocomplexes are observed (Ebong et al., 2011), and kinetic experiments show that the TPR domain containing peptidyl-

prolyl-isomerase (PPIase) Cpr6 partially displaces Sti1 from a Hsp90:Sti1 2:2 complex, suggesting that heterocomplexes are favored (Li et al., 2011). Further addition of Aha1 leads to full displacement of Sti1 but does not displace Cpr6. Aha1 and Cpr6 synergistically stimulate Hsp90’s ATPase activity, suggesting that both cochaperones favor the closed N-terminally dimerized conformation, which disfavors Sti1 binding. Aha1, in turn, is displaced by p23/Sba1, which also stimulates dissociation of Cpr6 in the presence of AMPPMP (Li et al., 2011; 2013). Although p23/Sba1 was crystallized in a symmetric 2:2 complex with Hsp90 (Ali et al., 2006), isothermal titration calorimetry demonstrated a 1:2 binding stoichiometry, and 2:2 binding may only occur at very high concentrations (Siligardi et al., 2004). Therefore, p23/Sba1 either competes directly with the C-terminal domain of Aha1 for binding to the same face of the dimerized NTDs or interacts with the opposite face and changes the conformation of the NTDs and maybe the MDs of the Hsp90 dimer to decrease the affinity for Aha1. In addition, p23/Sba1 has a strong rectifying effect on the conformational cycle of Hsp90 (Ratzke et al., 2014). P23/Sba1 preferentially binds to the ATP-bound closed conformation and dissociates with higher rates from the nucleotide-free closed conformation. In addition, p23/Sba1 inhibits ATPase activity of Hsp90 by slowing down product release (Graf et al., 2014) and is proposed to thus stabilize the Hsp90 client complex in the closed conformation (Prodromou et al., 2000). The kinase-specific cochaperone Cdc37 also seems to favor asymmetric complexes with Hsp90 when clients are present (see below, Vaughan et al., 2006). Asymmetry in Hsp90 cochaperone complexes can also be induced by an asymmetric conformation of a dimeric cochaperone, as in the case of the E3 ligase CHIP, which only interacts with a single MEEVD motif at the C terminus of Hsp90, despite having two complete TPR domains per dimer, and links Hsp90 to single Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc. 11

Molecular Cell

Review ATP

A-NT A-NTD B’-NTD B’-N

A

NTD

NTD CL C CL

MD 30°

MD CTD

A-M A-MD B’-MD B’-

CL CTD

Sgt1 Aha1 Sba1 Cdc37 Aha1+Sba1 Sba1+Cdc37

Sti1

B

B 30°

A A-CTD B’-CTD B TAU

GR

TAU+GR

p53

∆131∆

C Y197

Figure 3. Asymmetric Structure of TRAP1

Y627

Cartoon representation of the crystal structure of Danio rerio TRAP1 (PDB ID: 4IPE; Lavery et al., 2014) with chain B duplicated, in light gray as in the structure and colored (B’-NTD, greencyan; B’-MD, cyan; B’-CTD, deep teal) overlaid to chain A (A-NTD, yellow; A-MD, orange; A-CTD, dark red) to illustrate the asymmetry of the TRAP1 dimer. Arrows indicate how the MD of A is moved out of the symmetric position. For the overlay, NTDs and CTDs were aligned. See Movie S1.

ubiquitin-conjugating E2 enzymes in an asymmetric conformation (Zhang et al., 2005). Alternatively, a cochaperone may contain two TPR domains like human Spagh, both of which interact with an MEEVD motif of the Hsp90 dimer, thus creating asymmetry linkage to Hsp90 clients (Pal et al., 2014). Taken together, the 1:2 asymmetric complexes, in which cochaperone binding to one protomer affects the conformation of both protomers in the Hsp90 dimer, provide the basis for an ordered succession of cochaperones and for a unidirectional conformational cycle of Hsp90 (Figure 2). Such a stoichiometry is also favored in vivo because the concentration of Hsp90 is much higher than the concentration of any one of its cochaperones (Figure 5) (Finka and Goloubinoff, 2013; Ghaemmaghami et al., 2003). Asymmetry in Hsp90-cochaperone interaction may also serve for recruiting single clients to the Hsp90 dimer. Whether the asymmetry in the cochaperone complexes extends in every case to conformational differences between the two protomers as observed for TRAP1 and to a sequential order of ATP hydrolysis is not clear. Chaperoning of Client Proteins and Interplay between Hsp70 and Hsp90 Machineries The modalities of interaction between Hsp90 and its client proteins are not well understood. Although many proteins may interact transiently with Hsp90, a limited set of proteins, with protein kinases and transcription factors making a significant 12 Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc.

Y313 Y627

Sp+Tp

Yp

Ac

Y197

30°

Ac+Ub

m1+Ub

Ac+Ub+Sm

S-NO

Figure 4. Interaction Surfaces and Posttranslational Modifications in Hsp90 (A–C) Surface representations of homology models of human Hsp90a onto the crystal structure of yeast Hsp82 (PDB ID: 2CG9; Ali et al., 2006; left panels) and E. coli HtpG (PDB ID: 2IOQ; Shiau et al., 2006; right panels) (Arnold et al., 2006; Biasini et al., 2014). Protomers colored in light and dark gray with residues involved in cochaperone binding (A), client binding (B), or posttranslationally modified (C) are highlighted as indicated. (A) We colored residues directly involved in the interaction of Hsp90 with cochaperones as demonstrated by crystallography (Aha1, PDB ID: 1USU, Meyer et al., 2004; Sba1, PDB ID: 2CG9, Ali et al., 2006; Cdc37, PDB ID: 1US7, Roe et al., 2004; Sgt1, PDB ID: 1RL1, Zhang et al., 2008) or implicated in interaction by peak broadening or chemical shift perturbation in NMR experiments (Aha1, Retzlaff et al., 2010; Sti1, Schmid et al., 2012) or protection in hydrogen exchange experiments (Sti1, Lee et al., 2012). Several residues are implicated in binding to more than one cochaperone (colored in purple and dark teal). (B) We colored residues of Hsp90 implicated in interaction with clients by peak broadening or chemical shift perturbation in NMR experiments, by SAXSderived models, or by hydrogen exchange data (TAU, courtesy of S. Ru¨diger, Karago¨z et al., 2014; GR, courtesy of L. Freiburger, Lorenz et al., 2014; p53, Hagn et al., 2011; staphylococcal nuclease D131D, Street et al., 2012). (C) We colored residues modified by posttranslational modifications (www. phosphosite.org). Sites only found in a single high-throughput mass spectrometry study are not colored. Lysines only found to be ubiquitinated were also left away for clarity. Right protomer in cartoon representation with sidechains modified posttranslationally as spheres, illustrating the density of posttranslational modifications. Indicated are phosphorylation sites mentioned in the review.

Molecular Cell

Review A

B

fraction of it, spend a considerable part of their lifetime in Hsp90containing complexes. The different groups of these bona fide Hsp90 clients share neither structural features nor sequence motifs. In contrast, the intrinsic thermodynamic stability seems to be the main criterion for affinity and interaction half-life, with stronger binding being observed with increasing unfolding propensity (Taipale et al., 2012). Therefore, typical Hsp90 clients may arrest folding at a late stage in the folding pathway to await upstream signals like phosphorylation for activation (e.g., protein kinases, some transcription factors), insertion of specific hydrophobic ligands into a large internal pocket (e.g., steroid hormone receptors, heme-activated transcription factor), or interaction with regulatory subunits (e.g., cyclin-dependent kinases) or may exist in dynamic equilibrium between alter-native conformations. Many attempts were undertaken to localize the client binding site to a specific domain of Hsp90 yielding different results (reviewed in Ro¨hl et al., 2013). The first clear picture was provided by a cryo-EM study of the complex of Hsp90 with Cdc37 and Cdk4, showing Cdk4 bound to NTD and MD (Vaughan et al., 2006). Recent structural investigations on a model substrate, a fragment of staphylococcal nuclease called D131D, and the bona fide clients glucocorticoid receptor ligand binding domain and Tau shed more light onto this mechanistically important issue. Chaperoning of the Model Substrate D131D The D131D fragment of staphylococcal nuclease is a monomeric largely unfolded protein with a small central hydrophobic core and local native-like structure and the fortunate property of relatively low aggregation propensity, which permitted its extensive characterization by NMR. Combining SAXS, FRET, and NMR measurements provided compelling evidence that E. coli Hsp90 binds with significant affinity to the locally structured region of D131D. It does not bind to D131D when driven to the fully folded conformation by interaction with a tight binding inhibitor and binds only weakly to the completely unstructured parts of the apo protein (Street et al., 2011; 2012). These results suggest that Hsp90 selects from conformational ensembles the partially folded molecules. This hypothesis was substantiated by use of full-length staphylococcal nuclease, which is generally well folded but rarely samples a more expanded partially unfolded state. Only the subset of molecules in the more unfolded state

Figure 5. Hsp90 Cochaperones (A) Cochaperones binding simultaneously to Hsp90 during maturation of steroid hormone receptors, kinases, or other Hsp90 clients. (B) Relative abundance of Hsp90 (Hsp82 and Hsc82) and cochaperones in yeast (Ghaemmaghami et al., 2003).

was recognized by E. coli Hsp90 (Street et al., 2014), reminiscent of previous observations for p53 (Ru¨diger et al., 2002). In the absence of nucleotide, D131D seems to bind to the inner MD face of the Hsp90 dimer and stabilizes a partially closed V-shape conformation, possibly interacting with both Hsp90 protomers, resulting in a substrate-induced compaction of Hsp90 (Street et al., 2011). Such a compaction could be achieved by D131D first binding and subsequently driving the conformational transition. Alternatively, D131D could select the compact Hsp90 conformation out of the ensemble of rapidly interconverting conformations. The second scenario seems to be the case because an Hsp90 amino acid replacement variant that is mostly in the wide-open conformation does not bind to D131D, and the conformation of this variant is also not affected by it (Street et al., 2014). D131D also accelerates AMPPNP-induced transition to the fully closed conformation and consequently stimulates the weak ATPase activity of E. coli Hsp90. In the presence of AMPPNP, D131D interacts with the Hsp90 dimer differently as compared to the apo state and with higher affinity. It seems to bind to the exposed surface of the MD of a single protomer, but also has transient contacts to the trans protomer (Street et al., 2011; 2012). It also influences the conformational dynamics of the NTD and NTD-MD rotation. Interestingly, experiments with wild-type mutant heterodimers revealed that binding of D131D to one protomer influences the NTD-MD rotation of the other protomer reminiscent of the above-mentioned effect of Aha1. The general idea emerging from these studies is that Hsp90 adapts its conformation to match the interaction sites offered by the client and interacts with transiently unfolding clients from a pool of folded ones. Chaperoning of the Glucocorticoid Receptor The steroid hormone receptors, in particular glucocorticoid receptor (GR) and progesterone receptor, belong to the first proteins recognized as obligate Hsp90 clients and served as paradigm for a maturation and activation cycle that requires Hsp40, Hsp70, Hop, Hsp90, and p23 as minimal chaperone machinery (Morishima et al., 2000). However, the instability and high aggregation propensity of this family of proteins precluded in vitro studies with purified proteins at concentrations necessary for biophysical and structural investigations. Two recent studies addressed the issue of chaperoning the glucocorticoid receptor (GR), using only the essential part for interaction with the chaperones, the ligand-binding domain (LBD), which in addition was stabilized by mutations and/or fusion to maltose binding protein for increased solubility, and shed light on binding, Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc. 13

Molecular Cell

Review activation, and concerted actions of Hsp90, Hsp70, and their cofactors (Kirschke et al., 2014; Lorenz et al., 2014). Binding of GRLBD to Hsp90 was nucleotide dependent, suggesting a preference for a moderately open V-shaped conformation over the closed ATP hydrolysis competent state, consistent with the observation that ATP hydrolysis promotes client dissociation (Young and Hartl, 2000). In contrast to observations for D131D, binding of GRLBD slowed down the transition of Hsp90 from the open to the closed state in the absence of cochaperones, decreasing the ATPase rate and consequently increasing the dwell time of the client on the chaperone. D131D might represent a folding intermediate that binds in a more extended conformation to both protomers of the Hsp90 dimer. Hsp90’s transitions between open and closed conformations might aid compaction of the folding intermediate, which in reverse induces in the Hsp90 dimer a bias toward the closed conformation, resulting in stimulation of the ATPase activity and subsequent release of the client. GR might be an example for clients that are moved in complex with Hsp90 to specific locations within the cell (Pratt et al., 1999). For GR, it was shown that after activation by hormone binding it remains associated with Hsp90 and travels to the nucleus (Harrell et al., 2004). It might be more advantageous for such clients to inhibit Hsp90’s ATPase activity to prevent premature release. They therefore might prefer to bind and stabilize more open conformations of Hsp90. The binding site for the GRLBD is mainly located in the MD of Hsp90 with some contacts in NTD and CTD, overlapping largely with the binding site for Cdk4 (Vaughan et al., 2006), p53 (Hagn et al., 2011), D131D (Street et al., 2012), and Tau (Karago¨z et al., 2014) (Figure 4; see below). Incubation of GRLBD with Hsp70, Hsp40, and ATP lead to structural changes around its ligand binding pocket and to a loss of its hormone binding capability (Kirschke et al., 2014). Further addition of Hsp90, Hop, and p23 restored the hormone binding activity of GRLBD. In the presence of the complete chaperone system hormone association and dissociation rates were increased as compared to the rates in the absence of chaperones, resulting in 3-fold higher affinity of GRLBD for hormone and presumably in a reduced response time to changing hormone concentrations, switching the hormone signal fast on and fast off. Cryo-EM images of the GRLBD-Hsp70-Hop-Hsp90 loading complex shows Hsp90 in a V-shape half-open conformation with Hsp90’s NTDs clamping the nucleotide binding domain (NBD) of Hsp70 (Kirschke et al., 2014). The substrate binding domain of Hsp70 is found perpendicular to the plane defined by the Hsp90 V-shape and the NBD of Hsp70. The resolution of the EM images was not sufficient to unambiguously place Hop and GRLBD. The molecular details of how GRLBD-loaded Hsp70 enters this complex and how it transfers the client onto Hsp90 remains enigmatic. Chaperoning Kinases The largest coherent group of Hsp90 clients are kinases (Taipale et al., 2012). An electron microscopy (EM) study revealed a ternary complex containing Hsp90, Cdc37, and the protein kinase Cdk4 in a 2:1:1 stoichiometry (Vaughan et al., 2006). In this complex the Hsp90 dimer is not N-terminally dimerized, and the two protomers have different conformations. As com14 Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc.

pared to the crystal structure of the closed N-terminally dimerized conformation, one protomer deviates mainly in the orientation of the NTD relative to MD, with the NTD being rotated outward away from the NTD of the second protomer. The second protomer deviates in the orientation of MD relative to CTD leading to an enlarged distance between the C-terminal parts of the two MDs in the dimer. A single Cdc37 molecule is bound between the two NTDs of the Hsp90 dimer, and one Cdk4 binds to NTD and MD of the protomer in the more open conformation. Chaperoning of Tau Hsp90’s clients generally have significant secondary and tertiary structure. An exception is the microtubule binding protein Tau, which belongs to the class of intrinsically disordered proteins. Tau oligomerization and aggregation into amyloid fibrils is a major feature of Alzheimer’s disease, progressive supranuclear palsy, and other neurodegenerative diseases generally called tauopathies (Clavaguera et al., 2014). Thus, understanding the interplay between Tau and molecular chaperones is of major medical interest in aging societies. The role of Hsp90 in tauopathies is still poorly understood. Hsp90 was proposed to interact with hyperphosphorylated Tau and to restore its microtubule binding activity by facilitating Tau dephosphorylation by the Hsp90 cochaperone and phosphatase PP5 (Liu et al., 2005). Hsp90 was also shown to promote degradation of hyperphosphorylated Tau in cooperation with its cochaperone and E3 ligase CHIP (Dickey et al., 2007). Remarkably, inhibition of Hsp90 also leads to Tau degradation, a property observed for many bona fide clients of Hsp90 (Salminen et al., 2011). In a recent study, Ru¨diger and colleagues used NMR and SAXS to determine the Hsp90 binding site within the Tau sequence and the Tau binding site on the Hsp90 dimer (Karago¨z et al., 2014). Hsp90 interacts with the repeats region of Tau, which is also the microtubule binding site. This site is characterized not by a specific binding motif, but rather by scattered large hydrophobic and positively charged residues in higher abundance than the remainder of the Tau sequence, which, as other intrinsically disordered proteins, has an overall low abundance of large aliphatic and aromatic amino acids. In comparison, the Hsp70 binding site overlaps with the Hsp90 binding site but is much smaller and consists of the few larger clusters of hydrophobic residues. Interestingly, part of the Hsp70 and Hsp90 binding site is known to be involved in amyloid fibril formation. In respect to Hsp90, the Tau binding site is very broad, involving both NTD and MD. In this client-bound state, Hsp90 is in an open conformation, with the NTDs pointing in opposite directions. Thus, a network of hydrophobic and charged residues are scattered and form the extended 106 A˚ long binding site that seems to be designed for large substrates. In this conformation, known cochaperone binding sites remain free and thus simultaneous binding of client and cochaperones may occur. The Tau binding site partially overlaps with the binding sites for Cdk4, GR, and the model substrate D131D (Genest et al., 2013; Street et al., 2011; Vaughan et al., 2006). Addition of ATP to the preformed Hsp90Tau complex modulates Hsp90 conformational dynamics and most likely breaks the symmetry within the Hsp90 dimer, as indicated by splitting of some NMR signals. Ru¨diger and co-workers suggest that Tau, in contrast to intrinsically disordered proteins in general, is recognized as Hsp90 client because it shares

Molecular Cell

Review specific features of late folding intermediates in which large hydrophobic clusters are already buried in the core of the protein and scattered small hydrophobic sites may still be exposed. Based on these results, they propose a general model for the interplay between the Hsp70 and Hsp90 machineries. First, Hsp70 interacts with big hydrophobic clusters exposed in nascent polypeptide chains. Dynamic binding and release of these sites allows rearrangements and condensation of the polypeptide, which buries Hsp70 binding sites, leaving scattered hydrophobic residues exposed that can interact with the extended client-binding site of Hsp90. Late folding by Hsp90 then buries those scattered residues, leading to the release of the fully matured client. Regulation of Hsp90 by Posttranslational Modifications As elaborated above, ATP binding and hydrolysis and interaction with cochaperones regulate Hsp90’s chaperone activity by modifying its conformational equilibria and transitions between different conformational states and by driving progression through its ATPase cycle. To this intricate chaperone cycle another layer of complexity is added by posttranslational modifications, including phosphorylation and acetylation at multiple sites, S-nitrosylation, methylation, ubiquitination, and sumoylation, which are spread over all three domains of Hsp90 and overlap with client and cochaperone binding sites (Figure 4) and bias certain conformations and alter interaction with cochaperones, thus fine-tuning Hsp90’s chaperone activity. Phosphorylation: Tuning Cycle Progression Biochemical and phosphoproteomics studies revealed so far 23/28 serine, 25/19 threonine, and 14/14 tyrosine residues in human Hsp90a and b, respectively. The physiological impact of phosphorylation has only been studied for few of these sites to some extent. As a general rule, replacement of the phosphorylated residue by glutamate or aspartate has a negative effect on Hsp90’s function, as it affects the chaperone’s ATPase activity and its conformational dynamics as well as the interaction with cochaperones and client proteins (Mollapour et al., 2010a; 2010b). However, certain clients, in particular protein kinases, depend on phosphorylation of Hsp90 for attaining their active state. Thus, the dynamics of phosphorylation and dephosphorylation contribute to the tuning of Hsp90’s activity and to client specificity (Miyata, 2009; Mollapour et al., 2010b; 2011; Zhao et al., 2001). Interestingly, Hsp90 can be phosphorylated by its own client proteins, like the kinases B-Raf, Akt, CK2, and PKCg (see below), and dephosphorylated by the protein phosphatase PP5/Ppt1, a TPR domain-containing cochaperone of Hsp90, which also dephosphorylates Hsp90’s cochaperone Cdc37 (Barati et al., 2006; Vaughan et al., 2008; Wandinger et al., 2006). Some phosphorylation sites are conserved between Hsp90s, whereas some are isoform specific. In an extensive mass spectrometric analysis, Buchner and colleagues identified several phosphorylation sites in NTD and MD of yeast Hsp90, including two sites targeted specifically by the cochaperone Ppt1 (Ser485 and 604) (Soroka et al., 2012). In vivo and in vitro experiments suggested that phosphorylation at these sites occurs in vivo only transiently and phosphorylation is not essential under the condition tested. Most phosphomi-

metic Hsp90 variants exhibited a decreased ATPase activity, and two of them were less responsive to inhibition of the ATPase activity by p23/Sba1 and, more importantly, the stimulation of the ATPase activity by Aha1. Thus, phosphorylation influences progression through the conformational cycle and fine-tunes the regulation by cochaperones. It remains to be elucidated under which conditions and at which stage of the chaperone cycle the individual sites are phosphorylated and dephosphorylated; whether phosphorylation and dephosphorylation are client specific, influenced by the client bound, or a precondition for binding-specific clients; and how these phosphorylation-induced changes of the conformational cycle affect client maturation. PKCg is one example of a kinase client of Hsp90 actively involved in its own regulation via a ‘‘phosphorylation switch’’ (Lu et al., 2014). Hsp90 and its kinase-specific cochaperone Cdc37 interact with PKCg, thus inhibiting its degradation and promoting its activation and translocation to the membrane. PKCg phosphorylates Hsp90 at three threonine residues, leading to a decrease of ATP binding and hydrolysis and reduced affinity for Cdc37 and, interestingly, for the PKCg client itself. Therefore, partially folded, inactive PKCg mediated by Cdc37 may bind to the unphosphorylated Hsp90. The chaperone cycle of Hsp90 assists maturation and membrane translocation of PKCg, which upon activation by Ca2+ and diacylglycerol phosphorylates Hsp90 in cis to provoke its timely release. Alternatively, or in addition, high concentrations of active PKCg may phosphorylate Hsp90 in trans, thus regulating activation of newly synthesized PKCg in a negative feedback loop. An ordered succession of phosphorylation and dephosphorylation of Hsp90 and its cochaperones at multiple sites during progression through the chaperone cycle seems to be a more general principle (Figure 6) (Xu et al., 2012). Phosphorylation of Cdc37 at Ser13 by CK2 promotes efficient binding to kinase clients and interaction with Hsp90 (Miyata and Nishida, 2004; Vaughan et al., 2008); subsequent recruitment of the TPRdomain containing phosphatase PP5/Ppt1 leads to dephosphorylation of Cdc37. Then, phosphorylation of Cdc37 at Tyr298 (for certain kinase clients also at Tyr4) and of Hsp90 at Tyr197 by the Yes kinase induces conformational changes in Hsp90 and accelerates dissociation of Cdc37 (Xu et al., 2012). A second phosphorylation of Hsp90 at Tyr313 promotes progression along the chaperoning cycle by inducing additional structural rearrangements favorable for recruitment of Aha1. Finally, the phosphorylation of Hsp90 at Tyr627 contributes to the release of Aha1, PP5/Ppt1, and the activated client kinase. Starting of a new cycle likely involves dephosphorylation of Cdc37 and Hsp90 by phosphatases that have not been identified yet. Thus, the succession of phosphorylation and dephosphorylation imprints directionality on the thermal conformational fluctuations of Hsp90. Whether simultaneous phosphorylation of both protomers of the Hsp90 dimer is necessary for this cycle or whether asymmetry is also dominating these processes has not been investigated. But in the view of the asymmetric binding of the cochaperones, differential phosphorylation of the Hsp90 protomers seems sufficient to accelerate asymmetric association and dissociation of the cochaperones and may enhance directionality of the chaperone cycle. Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc. 15

Molecular Cell

Review P

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Figure 6. Regulation of the Progression through the Hsp90 Chaperone Cycle by Sequential Phosphorylation Phosphorylation on Cdc37 regulates its affinity for kinase clients (S13, positive; Y298, negative), and phosphorylation of Hsp90 regulates affinity for Cdc37 (Y197, negative) and Aha1 (Y313, positive; Y627, negative) (Xu et al., 2012). It is unclear whether both protomers of Hsp90 need to be phosphorylated.

Acetylation: Tuning Client Specificity As by phosphorylation, Hsp90 is regulated by the dynamics of acetylation and deacetylation, involving acetyltransferases like p300 and deacetylases of the HDAC family (Bali et al., 2005; Kovacs et al., 2005; Yu et al., 2002). Likewise, acetylation seems to have mostly a negative impact on the binding and maturation of client proteins, since HDAC inhibition leads to hyper-acetylated Hsp90 accompanied by reduced ATPase activity and defects in Hsp90-client complex formation (Bali et al., 2005). A large number of acetylation sites have been identified mostly by high-throughput mass spectrometry techniques (Figure 4), but only few of them were functionally characterized (Scroggins et al., 2007; Yang et al., 2008). Acetylation of specific sites might not only negatively impact Hsp90 activity, as suggested by certain acetyl-mimetic variants of human Hsp90a, which bound to the cochaperone CHIP and the client c-Raf more tightly than the non-acetylatable counterpart (Yang et al., 2008). Thus, transient acetylation could be important for interaction with certain cochaperones and chaperoning of some clients. Acetylation and deacetylation like phosphorylation may bias the thermal fluctuations-induced transitions of Hsp90 between different conformations, thus affecting the ATPase cycle and selection of cochaperones and clients. Interestingly, HDAC6, a major deacetylase of Hsp90, depends on its ubiquitin binding domain for interaction with and deacetylation of Hsp90 (Kovacs et al., 2005), and many of the acetylation sites were also found by mass spectrometry studies to be ubiquitinated, suggesting a complex interplay between these competing modifications on Hsp90. 16 Molecular Cell 58, April 2, 2015 ª2015 Elsevier Inc.

SUMOylation: Accelerating the Chaperone Cycle The small ubiquitin-like modifier SUMO is activated like ubiquitin and linked to substrate proteins posttranslationally via an isopeptide bond to the ε-amino group of a lysine residue. In general, SUMOylation modifies the activity of the target protein by altering its conformation, localization, or interaction with co-factors (Flotho and Melchior, 2013). Mollapour and colleagues identified a conserved lysine (K178 in yeast, K191 in human) as a SUMOylation site (Mollapour et al., 2014). This residue is not accessible in the closed crystal structure, suggesting SUMOylation occurs during the conformational cycle prior to NTD dimerization and dimer closure. Though, ATP binding is a pre-requisite for SUMOylation and subsequent Aha1 binding. In vivo, SUMOylation of K178 on one Hsp90 protomer is required and sufficient to recruit Aha1 but has no influence on the interaction with other cochaperones like p23/Sba1, Hop/Sti1, or Cdc37. This asymmetric SUMOylation fits well to the previously described asymmetric recruitment of one Aha1 per Hsp90 dimer and to the asymmetry in ATP hydrolysis within the Hsp90 dimer (Cunningham et al., 2008; Mishra and Bolon, 2014; Retzlaff et al., 2010). The non-SUMOylatable variants chaperoned some clients like the kinases Ste11 and v-Src and the glucocorticoid receptor with similar efficacy as wild-type Hsp90. To the contrary, increased Hsp90 SUMOylation compromised activation of these clients, possibly due to more efficient Aha1 recruitment, leading to an acceleration of the chaperone cycle and consequently a reduced dwell time of the client on Hsp90. SUMOylation was even more detrimental to maturation of the difficult-to-fold substrate

Molecular Cell

Review CFTR, which was more stable in the presence of the non-SUMOylatable variant than in the presence of wild-type Hsp90. The positive effect of the non-SUMOylated Hsp90 variant, which fails to recruit Aha1, is consistent with the earlier result of increased CFTR maturation upon knockdown of Aha1 (Koulov et al., 2010; Wang et al., 2006). In yeast, the degree of Hsp90 SUMOylation was not affected by heat shock or a-factor-mediated cell-cycle arrest. In contrast, treatment with ATP-competitive Hsp90 inhibitors increased the level of Hsp90 SUMOylation. Interestingly, these inhibitors also bound preferentially to SUMOylated Hsp90, likely displacing ATP and trapping the chaperone in an open-like conformation, thus blocking progression through the chaperone cycle. Moreover, Hsp90 SUMOylation levels are increased in transformed cells, providing an extra explanation for the greater sensitivity of cancer cells to Hsp90 inhibition (Mollapour et al., 2014; Trepel et al., 2010). Total Complexity of Posttranslational Modifications In addition to the 59/56 phosphorylation, 33/24 acetylation, the SUMOylation, and S-nitrosylation site, high-throughput proteomics approaches revealed 39/46 ubiquitination, 7/11 succination, 1/2 monomethylation, and 1/0 dimethylation sites in human Hsp90a and b, respectively (www.phosphosite.org). The functional relevance for most of these modifications has not been established rigorously. In addition, many modifications target the very same residues, e.g., K191 in human Hsp90a was shown to be SUMOylated, acetylated, and ubiquitinated. The complexity of this regulatory system exceeds current analysis capabilities. If all of these modifications are functionally relevant and independent of each other, human Hsp90a and Hsp90b exist in 3 3 1042 and 7 3 1041 different variants, numbers that are much larger than the total number of Hsp90 molecules in a human body (ca. 1021). In how many cases is Hsp90 modified by the very client it activates, as mentioned above for phosphorylation by PKCg or as shown for S-nitrosylation of human Hsp90a by nitric oxide synthase (Martı´nez-Ruiz et al., 2005; Retzlaff et al., 2009)? Conclusion and Outlook Asymmetry induced by intrinsic strain upon N-terminal dimerization or extrinsically by interaction with cochaperones or by posttranslational modifications seems to be a dominating feature of the homodimeric Hsp90 chaperones. This asymmetry imposes a bias on the conformational equilibria during progression through the chaperone cycle, guaranteeing an ordered succession of events. For client binding, Hsp90s present a large interaction surface spread over all three domains, which is characterized by scattered hydrophobic, hydrophilic, and charged patches, offering many low-affinity contact sites. The high conformational dynamics may be key to Hsp90’s chaperoning power, allowing adaptation to ‘‘soft’’ surfaces of highly dynamic folding intermediates or any protein that in its native state is highly dynamic and prone to transient unfolding and thus combines many characteristics of folding intermediates. They may also be a prerequisite for allowing rearrangements of the client in the course of the activation process. The different conformations accessed during the activation process may vary with different clients and, in fact, seem to be influenced by the client itself.

Future challenges include the elucidation of the structural features of client loading and transitions between different stages of client activation at the molecular level. This now seems feasible due to the recent revolution in cryo-EM technology, providing near-residue resolution images; thus, dynamic transient processes can be captured. These images together with other biophysical data may answer questions such as the following: what happens to the client when Hsp90 binds to it; how is it remodeled and what are the likely asymmetric conformational changes in the two Hsp90 protomers and in cochaperones that allow such a remodeling of the client; and how does client activation by ligand binding or modification by upstream signals affect Hsp90 to release the active client or to transport it to the site of action? The answers to these questions are likely different for each client. How these dynamic processes are further regulated by posttranslational modifications to fit the needs of the cell and as reaction to environmental and developmental cues will be another large field of future research. Elucidating the different subpopulations of specifically modified Hsp90s present in any given cell type will be crucial for defining the potential of Hsp90 in these cells to activate a defined set of clients and for understanding the role of Hsp90 in health and disease. SUPPLEMENTAL INFORMATION Supplemental Information includes Movie S1 and Movie S1 legend and references and can be found with this article online at http://dx.doi.org/10.1016/j. molcel.2015.02.022. REFERENCES Ali, M.M.U., 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-nucleotide-p23/Sba1 closed chaperone complex. Nature 440, 1013–1017. Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006). The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201. Bali, P., Pranpat, M., Bradner, J., Balasis, M., Fiskus, W., Guo, F., Rocha, K., Kumaraswamy, S., Boyapalle, S., Atadja, P., et al. (2005). Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors. J. Biol. Chem. 280, 26729–26734. Barati, M.T., Rane, M.J., Klein, J.B., and McLeish, K.R. (2006). A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells. J. Proteome Res. 5, 1636–1646. Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Cassarino, T.G., Bertoni, M., Bordoli, L., and Schwede, T. (2014). SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258. Clavaguera, F., Grueninger, F., and Tolnay, M. (2014). Intercellular transfer of tau aggregates and spreading of tau pathology: Implications for therapeutic strategies. Neuropharmacology 76 (Pt A), 9–15. Cunningham, C.N., Krukenberg, K.A., and Agard, D.A. (2008). Intra- and intermonomer interactions are required to synergistically facilitate ATP hydrolysis in Hsp90. J. Biol. Chem. 283, 21170–21178. Cunningham, C.N., Southworth, D.R., Krukenberg, K.A., and Agard, D.A. (2012). The conserved arginine 380 of Hsp90 is not a catalytic residue, but stabilizes the closed conformation required for ATP hydrolysis. Protein Sci. 21, 1162–1171. Dickey, C.A., Kamal, A., Lundgren, K., Klosak, N., Bailey, R.M., Dunmore, J., Ash, P., Shoraka, S., Zlatkovic, J., Eckman, C.B., et al. (2007). The high-affinity

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Harrell, J.M., Murphy, P.J.M., Morishima, Y., Chen, H., Mansfield, J.F., Galigniana, M.D., and Pratt, W.B. (2004). Evidence for glucocorticoid receptor transport on microtubules by dynein. J. Biol. Chem. 279, 54647–54654. Johnson, J.L. (2012). Evolution and function of diverse Hsp90 homologs and cochaperone proteins. Biochim. Biophys. Acta 1823, 607–613. Karago¨z, G.E., Duarte, A.M.S., Ippel, H., Uetrecht, C., Sinnige, T., van Rosmalen, M., Hausmann, J., Heck, A.J.R., Boelens, R., and Ru¨diger, S.G.D. (2011). N-terminal domain of human Hsp90 triggers binding to the cochaperone p23. Proc. Natl. Acad. Sci. USA 108, 580–585.

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Karago¨z, G.E., Duarte, A.M.S., Akoury, E., Ippel, H., Biernat, J., Mora´n Luengo, T., Radli, M., Didenko, T., Nordhues, B.A., Veprintsev, D.B., et al. (2014). Hsp90-Tau complex reveals molecular basis for specificity in chaperone action. Cell 156, 963–974.

Miyata, Y. (2009). Protein kinase CK2 in health and disease: CK2: the kinase controlling the Hsp90 chaperone machinery. Cell. Mol. Life Sci. 66, 1840– 1849.

Kirschke, E., Goswami, D., Southworth, D., Griffin, P.R., and Agard, D.A. (2014). Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles. Cell 157, 1685–1697.

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Molecular Cell

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Mollapour, M., Bourboulia, D., Beebe, K., Woodford, M.R., Polier, S., Hoang, A., Chelluri, R., Li, Y., Guo, A., Lee, M.-J., et al. (2014). Asymmetric Hsp90 N domain SUMOylation recruits Aha1 and ATP-competitive inhibitors. Mol. Cell 53, 317–329.

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