All-atomic molecular dynamic studies of human CDK8: Insight into the A-loop, point mutations and binding with its partner CycC

Abstract

The Mediator, a conserved multisubunit protein complex in eukaryotic organisms, regulates gene expression by bridging sequence-specific DNA-binding transcription factors to the general RNA polymerase II machinery. In yeast, Mediator complex is organized in three core modules (head, middle and tail) and a separable ‘CDK8 submodule’ consisting of four subunits including Cyclin-dependent kinase CDK8 (CDK8), Cyclin C (CycC), MED12, and MED13. The 3-D structure of human CDK8-CycC complex has been recently experimentally determined. To take advantage of this structure and the improved theoretical calculation methods, we have performed molecular dynamic simulations to study dynamics of CDK8 and two CDK8 point mutations (D173A and D189N), which have been identified in human cancers, with and without full length of the A-loop as well as the binding between CDK8 and CycC. We found that CDK8 structure gradually loses two helical structures during the 50-ns molecular dynamic simulation, likely due to the presence of the full-length A-loop. In addition, our studies showed the hydrogen bond occupation of the CDK8 A-loop increases during the first 20-ns MD simulation and stays stable during the later 30-ns MD simulation. Four residues in the A-loop of CDK8 have high hydrogen bond occupation, while the rest residues have low or no hydrogen bond occupation. The hydrogen bond dynamic study of the A-loop residues exhibits three types of changes: increasing, decreasing, and stable. Furthermore, the 3-D structures of CDK8 point mutations D173A, D189N, T196A and T196D have been built by molecular modeling and further investigated by 50-ns molecular dynamic simulations. D173A has the highest average potential energy, while T196D has the lowest average potential energy, indicating that T196D is the most stable structure. Finally, we calculated theoretical binding energy of CDK8 and CycC by MM/PBSA and MM/GBSA methods, and the negative values obtained from both methods demonstrate stability of CDK8-CycC complex. Taken together, these analyses will improve our understanding of the exact functions of CDK8 and the interaction with its partner CycC.

A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

“…Furthermore, the activation of CDKs requires the binding of Cyclins, and phosphorylation of THR, SER, TYR on their activation loop [57,58]. The binding of CycC changes the conformation of CDK8 dramatically [58,28], and enables the ligand binding to the allosteric binding [25]. This agrees with our observation that when CycC is absent, the αC helix of CDK8 adopts an αC-out conformation, in which the GLU66 moves away from the DMG motif.…”

“…Wu Xu, et. al presented 50 ns of all-atomic molecular dynamic studies of human CDK8 and provided insights into two point mutations D173A and D189N within the activation loop by the hydrogen bond (H-bond) dynamic study of the activation loop residues and the MMPB/SA method [28].…”

A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

“…The activation of CDKs requires the binding of cyclins and phosphorylation of Thr, Ser, and Tyr on their activation loop [63, 64]. This binding changes the conformation of CDK8 markedly [32, 64] and enables ligand binding in the allosteric site [25], which is supported by our observation that in the absence of CycC, the αC helix of CDK8 adopts an αC-out conformation, whereby Glu66 moves away from the DMG motif. By losing the H-bond from Glu66 and interactions from the entire αC helix, the allosteric binding site collapses, thereby disabling the binding of type-II ligands.…”

“…Callegari and coworkers ranked the residence time of a series of CDK8 type-II inhibitors using metadynamics and the ranking was roughly consistent with experimental data [31]. Xu et al, with 50 ns of all-atom MD studies of human CDK8, provided insights into two-point mutations, D173A and D189N, within the activation loop by using hydrogen bond (H-bond) dynamic study of the activation loop residues and the MM/PBSA method [32]. …”

Transient states and barriers from molecular simulations and milestoning theory: kinetics in ligand-protein recognition and compound design

“…40 A series of CDK8 drug candidates was discovered by structure-based drug design and virtual screening. 41,42 A series of pyrazolourea ligands (PLs) have been developed and targeted the allosteric binding site of CDK8, 43 and a few studies have used MD simulations to examine the structural stability and ligand binding of the CDK8/CycC complex 44,45 A recent work used a metadynamics-based protocol to successfully rank the experimental residence time of CDK8 inhibitors 46 . The screening method provides a tool to identify inhibitors with relative short or long residence time; however, further investigation to understand determinants with atomistic details that govern the binding kinetics is needed.…”