Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase

Verba, Kliment A.; Wang, Ray Yu-Ruei; Arakawa, Akihiko; Liu, Yanxin; Shirouzu, Mikako; Yokoyama, Shigeyuki; Agard, David A.

doi:10.1126/science.aaf5023

Cited by 386 publications

(548 citation statements)

References 56 publications

(41 reference statements)

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“…In general, the conformational landscape of the kinase is reshaped by the presence of the oncogenic mutations, which led to a less compact structure of regions that may specifically interact with Hsp90-Cdc37, especially the β1-β5-strands and the P-loop, supporting previous studies that suggested a correlation between Hsp90 binding and the openness of the kinase fold174041. Moreover, in the pY416-activated state of c-Src3MΔC, we found an even more extended kinase structure that may increase the interaction with Hsp90 and Cdc37.…”

Section: Discussionsupporting

confidence: 80%

Hsp90 dependence of a kinase is determined by its conformational landscape

Luo

Boczek

Wang

et al. 2017

Sci Rep

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Heat shock protein 90 (Hsp90) is an abundant molecular chaperone, involved in the folding and activation of 60% of the human kinome. The oncogenic tyrosine kinase v-Src is one of the most stringent client proteins of Hsp90, whereas its almost identical homolog c-Src is only weakly affected by the chaperone. Here, we perform atomistic molecular simulations and in vitro kinase assays to explore the mechanistic differences in the activation of v-Src and c-Src. While activation in c-Src is strictly controlled by ATP-binding and phosphorylation, we find that activating conformational transitions are spontaneously sampled in Hsp90-dependent Src mutants. Phosphorylation results in an enrichment of the active conformation and in an increased affinity for Hsp90. Thus, the conformational landscape of the mutated kinase is reshaped by a broken “control switch”, resulting in perturbations of long-range electrostatics, higher activity and increased Hsp90-dependence.

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Section: Discussionsupporting

confidence: 80%

Hsp90 dependence of a kinase is determined by its conformational landscape

Luo

Boczek

Wang

et al. 2017

Sci Rep

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“…iii) In addition to a decreased steady-state level of the Hsc82 (Hsp90) protein in naa10Δ cells (see item ii above), one would also expect the impairment of the Hsp90 system as a whole in the absence of NatA. The functioning of Hsp90 involves its transient binding to cochaperones, with some of them carrying bound clients (12). Given the role of Nt-Ac groups in modulating (usually enhancing) protein interactions within cognate protein complexes (49), decreased affinities among most components of the Hsp90 system, which are not Nt-acetylated in naa10Δ cells, would be expected to perturb both the delivery of clients to Hsp90 by cochaperones and other aspects of this circuit, based on transient protein interactions.…”

Section: Discussionmentioning

confidence: 99%

“…Cochaperones of the ∼90-kDa Hsp90 comprise, in mammals, ∼30 distinct proteins. They participate in the delivery of clients to Hsp90 and contribute to related processes, including ATP hydrolysis by Hsp90 (12). Because of its ability to modulate protein conformations, the Hsp90 system can either exacerbate or counteract the fitness-reducing effects of mutations in cellular proteins.…”

mentioning

confidence: 99%

Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

Hyun

Varshavsky

2017

Proc. Natl. Acad. Sci. U.S.A.

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We found that the heat shock protein 90 (Hsp90) chaperone system of the yeast Saccharomyces cerevisiae is greatly impaired in naa10Δ cells, which lack the NatA N α -terminal acetylase (Nt-acetylase) and therefore cannot N-terminally acetylate a majority of normally N-terminally acetylated proteins, including Hsp90 and most of its cochaperones. Chk1, a mitotic checkpoint kinase and a client of Hsp90, was degraded relatively slowly in wild-type cells but was rapidly destroyed in naa10Δ cells by the Arg/N-end rule pathway, which recognized a C terminus-proximal degron of Chk1. Diverse proteins (in addition to Chk1) that are shown here to be targeted for degradation by the Arg/N-end rule pathway in naa10Δ cells include Kar4, Tup1, Gpd1, Ste11, and also, remarkably, the main Hsp90 chaperone (Hsc82) itself. Protection of Chk1 by Hsp90 could be overridden not only by ablation of the NatA Nt-acetylase but also by overexpression of the Arg/N-end rule pathway in wild-type cells. Split ubiquitin-binding assays detected interactions between Hsp90 and Chk1 in wild-type cells but not in naa10Δ cells. These and related results revealed a major role of Nt-acetylation in the Hsp90-mediated protein homeostasis, a strong up-regulation of the Arg/N-end rule pathway in the absence of NatA, and showed that a number of Hsp90 clients are previously unknown substrates of the Arg/N-end rule pathway.roteins called chaperones promote the in vivo protein folding and counteract misfolding, maladaptive protein aggregation, and other perturbations of proteostasis (1-4). Hsp90 is an ATPdependent chaperone system that mediates the conformational maturation and stabilization of many proteins. Referred to as Hsp90 clients, these proteins include kinases, transcriptional regulators, and many other proteins that comprise at least 20% of a cellular proteome. The diversity of Hsp90 clients and the broad range of processes that involve Hsp90 underlie its necessity for cell viability in all eukaryotes (5-11). Cochaperones of the ∼90-kDa Hsp90 comprise, in mammals, ∼30 distinct proteins. They participate in the delivery of clients to Hsp90 and contribute to related processes, including ATP hydrolysis by Hsp90 (12). Because of its ability to modulate protein conformations, the Hsp90 system can either exacerbate or counteract the fitness-reducing effects of mutations in cellular proteins. This property of Hsp90 affects the standing genetic variation and thereby influences the impact of natural selection on rates and directions of evolution (13,14).The N-end rule pathways comprise a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, thereby causing the degradation of these proteins by the proteasome or autophagy in eukaryotes and by the proteasome-like ClpAP protease in bacteria (Fig. 1) (15-26). The main determinant of an N-degron is a destabilizing Nt-residue of a protein. Many N-degrons become active through their enzymatic Nt-acetylation, Ntformylation...

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“…While this article was in print the cryo-EM structure of the Hsp90-Cdc37-Cdk4 complex was published showing that Cdc37-phosphoSer13 contributes towards the coordination of a specific conformation of the Cdc37 N-terminal domain and its position relative to Hsp90 (39). This structure further supports our conclusion that a structural change is required for PP5-mediated dephosphorylation of Cdc37 in the context of the clientloaded Hsp90 chaperone complex and that this may be the trigger for client kinase release from the Hsp90-chaperone complex.…”

Section: Methodsmentioning

confidence: 99%

Structural and functional basis of protein phosphatase 5 substrate specificity

Oberoi

Dunn

Woodford

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone-and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper-or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.Hsp90 | PP5 | Cdc37 | chaperone | phosphatase P rotein phosphatase 5 (PP5) has pleiotropic roles in cellular signaling, including DNA damage repair, proliferation of breast cancer cells, circadian cycling, response to cytotoxic stresses, Rac-dependent potassium ion channel activity, and activation of steroid hormone receptors [e.g., glucocorticoid receptor (GR) and estrogen receptor] (1, 2). It is a member of the phosphoprotein phosphatase (PPP) family of serine/threonine phosphatases, which has members that share a highly conserved catalytic core and catalytic mechanism dependent on two metal ions, commonly Mn 2+ . Most PPP family members exhibit high, nonspecific phosphatase activity. Specificity is provided by a large cohort of regulatory and other interacting proteins that function to inhibit basal activity and recruit substrates, thereby finely tuning the enzymes (3). This combinatorial approach enables a small number of catalytic subunits to have the breadth of specificity equivalent to that seen in kinases, which are greater in number by an order of magnitude. Structures of complexes between regulatory and catalytic domains have illuminated the importance of regulatory subunits in facilitating substrate recruitment (3). However, to date, there is no structural information describing how a substrate binds at the active site of a PPP; therefore, a central question remains of how local interactions between the substrate and the catalytic domain contribute to the molecular basis of dephosphorylation.PP5 is unique among the PPP family because it has a low basal activity caused by an autoinhibitory N-terminal tetratricopeptide (TPR) domain (4). Its activity is promoted by a number of cellular factors, including fatty acids and the molecular chaperone heat shock protein 90 (Hsp90) (5), both of which release autoinhibition by interacting with the TPR...

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Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase

Cited by 386 publications

References 56 publications

Hsp90 dependence of a kinase is determined by its conformational landscape

Hsp90 dependence of a kinase is determined by its conformational landscape

Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

Structural and functional basis of protein phosphatase 5 substrate specificity

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