Abstract
Cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC) play conserved roles in modulating RNA polymerase II (Pol II)-dependent gene expression. To understand the structure and function relations of CDK8, we analyzed the structures of human and Drosophila CDK8 proteins using molecular dynamics simulations, combined with functional analyses in Drosophila. Specifically, we evaluated the structural differences between hCDK8 and dCDK8 to predict the effects of the LXXLL motif mutation (AQKAA), the P154L mutations, and drug binding on local structures of the CDK8 proteins. First, we have observed that both the LXXLL motif and the kinase activity of CDK8 are required for the normal larval-to-pupal transition in Drosophila. Second, our molecular dynamic analyses have revealed that hCDK8 has higher hydrogen bond occupation of His149-Asp151 and Asp151-Asn156 than dCDK8. Third, the substructure of Asp282, Phe283, Arg285, Thr287 and Cys291 can distinguish human and Drosophila CDK8 structures. In addition, there are two hydrogen bonds in the LXXLL motif: a lower occupation between L312 and L315, and a relatively higher occupation between L312 and L316. Human CDK8 has higher hydrogen bond occupation between L312 and L316 than dCDK8. Moreover, L312, L315 and L316 in the LXXLL motif of CDK8 have the specific pattern of hydrogen bonds and geometries, which could be crucial for the binding to nuclear receptors. Furthermore, the P154L mutation dramatically decreases the hydrogen bond between L312 and L315 in hCDK8, but not in dCDK8. The mutations of P154L and AQKAA modestly alter the local structures around residues 154. Finally, we identified the inhibitor-induced conformational changes of hCDK8, and our results suggest a structural difference in the drug-binding site between hCDK8 and dCDK8. Taken together, these results provide the structural insights into the roles of the LXXLL motif and the kinase activity of CDK8 in vivo.
Highlights
Cyclin-dependent kinase 8 (CDK8) and Cyclin C (CycC) are two conserved subunits of the transcription cofactor Mediator complex, which is involved in regulating most, if not all, of RNA polymerase II (Pol II)-dependent transcription in eukaryotes [1,2,3,4]
We have previously reported that ecdysone receptor (EcR) and USP can co-immunoprecipitate with CDK8, that CDK8 can directly interact with the EcR-activating function domain 1 (AF1) domain based on a yeast-two-hybrid assay, and that CDK8 has a highly conserved LXXLL motif [21]
Because the wild-type dCDK8 tagged with EGFP at the C-terminus of dCDK8 can rescue the cdk8 null mutant in the larval-pupal transition [21], we used the same approach and generated transgenic flies carrying a mutated version of the LXXLL motif, from LQKLL to AQKAA of dCDK8, as validated by sequencing (Figure 1a)
Summary
CDK8 and CycC are two conserved subunits of the transcription cofactor Mediator complex, which is involved in regulating most, if not all, of RNA Pol II-dependent transcription in eukaryotes [1,2,3,4]. Because CDK8 is the only enzymatic subunit of the Mediator complex, CDK8 can regulate Pol II-dependent gene expression by directly phosphorylating a number of transcription factors, including E2F1 [7,8], N-ICD (intracellular domain of Notch) [9], p53 [10], Smad proteins [11,12], SREBP (sterol regulatory element-binding protein) [13], and STAT1 (signal transducer and activator of transcription 1) [14,15,16]. Sci. 2020, 21, 7511 our structure comparison method can nearly accurately cluster proteins with CDK8 point mutations These results will advance our understanding of the structure-function relationships of CDK8 proteins
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