Atomic structure of Hsp90:Cdc37:Cdk4 reveals Hsp90 regulates kinase via dramatic unfolding

Abstract

The Hsp90 molecular chaperone and its Cdc37 co-chaperone help stabilize and activate over half of the human kinome. However, neither the mechanism by which these chaperones assist their client kinases nor why some kinases are addicted to Hsp90 while closely related family members are independent is known. Missing has been any structural understanding of these interactions, with no full-length structures of human Hsp90, Cdc37 or either of these proteins with a kinase. Here we report a 3.9Å cryoEM structure of the Hsp90:Cdc37:Cdk4 kinase complex. Cdk4 is in a novel conformation, with its two lobes completely separated. Cdc37 mimics part of the kinase Nlobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N-and C-lobe interfaces, protecting the kinase in a trapped unfolded state. Based on this novel structure and extensive previous data, we propose unifying conceptual and mechanistic models of chaperonekinase interactions. Main TextThe human kinome is responsible for regulating about a third of all proteins through phosphorylation(1). Proper regulation of this process is important, as misregulated kinase activity can lead to cell death and disease(2). To achieve fine regulation, kinase activity can be sensitively modulated by multiple allosteric inputs. Thus, kinase domains are organized so that dispersed small structural changes caused by binding of regulatory domains/proteins or phosphorylation, can significantly alter kinase activity. Examples of such regulator interactions abound, via SH2/SH3 domains for Src family kinases, dimerization for EGFR or Raf family kinases, and cyclin regulation for Cdks being well characterized examples(3). Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptBeyond these specific regulators, the Hsp90 molecular chaperone, a member of the general cellular protein folding machinery, also plays a fundamental role in the regulation of many kinases(4). While usually chaperones facilitate the early steps of protein folding, Hsp90 also functions late in the folding process to help both fold and activate a set of protein "clients" (~10% of the proteome)(5). Notably ~60% of the human kinome interacts with Hsp90 with the assistance of its kinase specific cochaperone Cdc37(6). Pharmocologic inhibition of Hsp90 leads to rapid ubiquitinylation and degradation of client kinases. As many Hsp90/ Cdc37-dependent kinases are key oncoproteins, (vSrc, bRafV600E, Her2, etc.) several Hsp90 inhibitors are undergoing clinical trials as cancer therapeutics(7).Hsp90 is a well conserved, but highly dynamic molecular machine. Each monomer within the Hsp90 dimer has three structural domains: a C-terminal domain (CTD) responsible for dimerization; a middle domain (MD) implicated in client binding; and the N-terminal domain (NTD) that binds ATP. Without bound nucleotide, Hsp90 mostly populates a variety of "open" states, whereas nucleotide binding promotes formation of...

Stimulation of heat shock protein 90 chaperone function through binding of a novobiocin analog KU-32

“…The modeled Hsp90␤ dimer structure (Fig. S6) was in complete agreement with its closed counterpart (30). Each of the four compounds (ATP, GA, NB, and KU-32) was docked, and their ⌬G of binding was calculated (Table S1).…”

Molecular Mechanism of Protein Kinase Recognition and Sorting by the Hsp90 Kinome-Specific Cochaperone Cdc37

“…Two cryoelectron microscopy (cryo-EM) structures (Vaughan et al, 2006; Verba et al, 2016) have shown that kinase domains interact with Hsp90 in an extended conformation, indicating that they undergo a large-scale structural rearrangement when in complex with Hsp90. However, Hsp90 does not form stable complexes with either bRaf or Cdk4 (Polier et al., 2013; Vaughan et al, 2006), despite the fact that the corresponding stable ternary Hsp90-Cdc37-kinase complexes can be formed.…”

“…It is plausible to assume that stable association of Cdk4 with Hsp90 occurs through a similar Cdc37-mediated conformational rearrangement of the kinase domain that involves partial unfolding. In this respect, the latest high-resolution cryo-EM structure of Hsp90-Cdc37-Cdk4 shows that the β 4 -β 5 strands and αC-β 4 loop of Cdk4 are completely unfolded, allowing the two kinase lobes to become separated (Verba et al, 2016). Notably, these structural elements partially overlap with the bRaf regions that expose unique proteolytic sites in complex with Cdc37 (Figures 4B and 4C; Supplemental Information) and, particularly, the αC-β4 loop and the beginning of β 4 strand.…”

Exploring the Functional Complementation between Grp94 and Hsp90

“…Finally, a recent cryo-electron microscopy study of a complex between protein kinase Cdk4, the cochaperone Cdc37, and human Hsp90 showed that the beta 5 strand of the kinase client is clamped between the two opposed middle domains of the Hsp90 dimer in the client-chaperone complex. The Cdc37 in this complex is also wrapped around the M 1 domain [61]. Together these data point to the essential role of the M 1 domain in client protein maturation.…”

“…Surprisingly, however, the grpN-hspMC chimera (4), which does not bind Cdc37 in our pulldown assay, nonetheless supports yeast viability. The Hsp90 binding site for Cdc37 was originally mapped to the N-terminal domain [42], but recent data from the cryo-EM structure of the Cdk4-Cdc37-hHsp90 complex [61] reveals that, in the context of the client assembly, Cdc37 makes the vast majority of its interactions with the M 1 domain of the chaperone ( Fig 7A ). Thus, although the grpN-hspMC chimera (4) lacks the cognate Hsp90 N-terminal domain, it does contain the alternate Hsp90 M 1 binding site for Cdc37 that is used in the context of client complexes.…”