Journal of Molecular Biology
Volume 412, Issue 2, 16 September 2011, Pages 251-266
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The Structure of CDK8/CycC Implicates Specificity in the CDK/Cyclin Family and Reveals Interaction with a Deep Pocket Binder

https://doi.org/10.1016/j.jmb.2011.07.020Get rights and content

Abstract

Cyclin-dependent kinase (CDK) 8 associates with cyclin C (CycC) and belongs to the CDK module of the Mediator of transcription, together with MED12 and MED13. CDK8 is involved in the regulation of mRNA transcription and was identified as a potent oncogene in colon cancerogenesis. We have solved the 2.2-Å crystal structure of CDK8/CycC in complex with sorafenib, an anti-cancer drug of clinical relevance. The CDK8 structure reveals a unique CycC recognition helix that explains the specificity of the CDK8/CycC pair and discrimination among the highly promiscuous binding in the CDK/cyclin family. In contrast to all CDKs, the CDK8 activation loop appears not to be phosphorylated. Based on the structure, we discuss an alternate mode of CDK8 activation to the general CDK activation by T-loop phosphorylation.

Sorafenib binds to the catalytic cleft of CDK8. It displays a deep pocket binding mode and is the first small molecule to induce a DFG-out conformation in the CDK family, which is actually DMG-out in CDK8.

Introduction

The hallmarks of cancer include insensitivity to anti-growth signals, leading to an uncontrolled, rather proliferative than quiescent, state of cell cycle.1 Cell cycle and transcription are tightly regulated by major players such as cyclin-dependent kinases (CDKs) and their regulatory cyclins.2, 3, 4 The physiological role of CDKs allows for classification, with the majority of CDK/cyclin pairs such as CDK2/cyclin A (CycA) and CDK4/cyclin D (CycD) being involved in the regulation of the cell cycle.5, 6, 7 At least three “C-terminal domain (CTD) kinases”—CDK7, CDK9, and CDK88—phosphorylate the CTD of RNA polymerase II (RNA Pol II), thus regulating transcription.9 The CDK8/cyclin C (CycC) complex belongs to the Mediator of transcription and forms the “CDK module” together with MED12 and MED13.10 CycC is promiscuous, as it also associates with CDK3. The CDK3/CycC pair accounts for cell cycle progression by phosphorylation of the retinoblastoma tumor suppressor RB.11, 12 The transcriptional CDK8/CycC complex suppresses transcription both directly, by phosphorylation of RNA Pol II in yeast,13 and indirectly, by phosphorylation of human cyclin H (CycH).14 In contrast, CDK8 has also been reported as a positive regulator of transcriptional gene activity (e.g. p53 target genes15 and the serum response network16), implying oncogenic effects. Analogously, the CDK module was postulated to be involved in the initiation and re-initiation of transcription.17 On top of that, CDK8 was identified as a potent oncogene in colon cancerogenesis involving β-catenin/wnt signaling.18, 19, 20 Thereby, CDK8 seems to function both Mediator-dependently and Mediator-independently.19 Recently, CDK8 was linked to epigenetics.21 In sum, these findings highlight the value of CDK8 as a drug target in cancer research. However, the development of selective inhibitors within the CDK/cyclin family is difficult, as the topology of the ATP binding site is very well conserved, generating promiscuity of ATP-competitive type I inhibitors.7 Alternatively, type II inhibitors extend from the ATP binding site into a neighboring pocket, the so-called “deep pocket.” The deep pocket is accessible by the rearrangement of the DFG motif from the active state (DFG-in) to the inactive state (DFG-out).22 However, CDK/cyclin complexes are not expected to allow the conformational changes in the ATP site that enable access to the adjacent deep pocket.23 Nevertheless, the DFG-out/deep pocket binder sorafenib (Nexavar®, BAY 43-9006)24, 25, 26 was shown to bind to CDK8/CycC.27

The CDK/cyclin family, especially CDK2 as monomer and in various complexes, has been structurally studied extensively. Further structures include human CDK4/CycD, CDK9/cyclin T (CycT), CDK5/p25, CDK6/cyclin Herpesvirus saimiri (vCyclin), CDK6/INK4s, CDK7, cyclin K, CycT/TAT/TAR, and CycH, as well as the yeast CycC analogue SRB11 (for a detailed overview, see Lolli6). A general mechanism for CDK activation and substrate specificity was based on the comprehensive structural information on CDK2 structures.28, 29 The binding of a cyclin to a CDK leads to a rearrangement of the αC helix towards the activation segment (αC helix pushed-in conformation). Then a reversibly phosphorylated residue of the T-loop interacts with a conserved arginine triad, maintaining the activation segment in an open conformation. However, further structural studies revealed alternate modes of CDK activation. A mechanism independent of activatory phosphorylation has been suggested for the CDK5/p25 pair.30 Interestingly, in the CDK4/CycD complex, association of the cyclin did not lead to an active kinase conformation of the αC helix.31, 32 An exception to a common CDK activation for CDK8 was pre-anticipated, as no phosphorylation of its activation segment was observed yet.

In contrast to cell cycle cyclins, which are tightly regulated by time-point-specific events, the level of transcriptional cyclins does not fluctuate during the cell cycle.6 This raises the question on how CDK8 gains further CycC specificity in contrast to CDK3, especially as CDK/cyclin formation in vitro is highly promiscuous. Since cyclins share a large common CDK binding surface, only subtle changes that improve the complementary shape and electrostatic charge of a CDK/cyclin pair lead to specificity.6 Hydrophobic contacts are central in the binding surface between any cyclin and any CDK.33

We have determined the 2.2-Å crystal structure of the human CDK8/CycC pair in complex with the inhibitor sorafenib. We show that an N-terminal helix, αB, which is unique to CDK8, makes extensive additional interactions with CycC besides the common binding surface. Its importance was underlined by truncation in a mutational analysis. It structurally explains discrimination against CDK3 and even other transcriptional CDK/cyclin complexes. Surprisingly, sorafenib binding reveals for the first time that it can induce a deep pocket binding mode with a DMG-out conformation in the CDK family.

The CDK8/CycC complex displays additional potential recognition surfaces for interactions, possibly for the recognition of MED12, MED13, or substrates.

Section snippets

The overall structure of CDK8/CycC

The overall CDK8/CycC architecture (Fig. 1a) is related to the previously solved CDK9/CycT and CDK2/CycA structures, where the binding mode of the transcriptional CDK9/CycT pair differs from that of the cell cycle complex CDK2/CycA by a relative rotation of the two components by 26°.34 We find an intermediate arrangement between these extreme positions for CycC when superimposing CDKs. CycC is rotated by about 10° along its H4 helix (Fig. 1b) relative to CycA, while ∼ 15° is still missing

Protein expression and purification

CDK8 and CycC templates were obtained by GENEART for subsequent PCR. Besides the CDK8 full-length construct, we evaluated C-terminally truncated construct variants (Hs1–464, Hs1–424, Hs1–403, Hs1–377, and Hs1–348) and a kinase dead mutant (Hs1–464 D173A). CDK8 constructs were cloned into the pFastBacHTa vector (Invitrogen), modified with the insertion of a glutathione S-transferase fusion with a PreScission protease cleavage site. CycC was cloned into the pFastBacHTa vector with additional

Acknowledgements

We thank Sabine Höppner for pointing out the relevance of this project and for indispensable discussions. We further thank Torsten Neuefeind, Dirk Ullmann, and Gerhard Müller for their continuous support, and other members of Proteros Biostructures GmbH for their scientific input. This work was financially supported by the Bundesministerium für Bildung und Forschung project BioChance PLUS-4 (Frag Screen) 0315161A.

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