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.

Keywords: CDK8; CycC; Molecular dynamics.

Figure 1. The sequence alignment of human and drosophila CDK8 and human CDK7 (PDB code: IUA2, A chain)

Sixteen amino acids in the middle of the human CDK8 A-loop are missing in the crystal structure of human CDK8 (PDB code: 3RGF, A chain).

Figure 2. The theoretical structure of human CDK8 based on molecular modeling and 50-ns molecular dynamics simulation

a, Change of potential energy as a function of time during the MD simulation; b, The theoretical structure with the lowest potential energy of human CDK8, which occurred at 49,408 ps during the MD simulation. The N-lobe, C-lobe, aB, aC, DMG motif, T196, A-loop and D189 are labeled; c, The backbone RMSD during the MD simulation is compared to the lowest potential energy conformation.

Figure 3. The molecular dynamic study of human CDK8

a, The CDK8 structure with the lowest potential energy during the 10-ns MD simulation; b, The CDK8 structure with the lowest potential energy during the 20-ns MD simulation; c, The CDK8 structure with the lowest potential energy during the 30-ns MD simulation; d, The CDK8 structure with the lowest potential energy during the 40-ns MD simulation.

Figure 4. Comparison of the CDK8 crystal and MD Structures

Cyan: crystal CDK8 structure; Orange: CDK8 MD structure

Figure 5. Dynamics of human CDK8 A-loop during the 50-ns molecular dynamic simulation

a, The two conformations of human CDK8 A-loop during the 50-ns MD simulation. The conformation with cyan color was from the structure with the lowest potential energy during the first 10-ns simulation, and the conformation with orange color was from the structure with the lowest potential energy during the 50-ns simulation; b, The conformation of the A-loop of human CDK8 crystal structure (PDB code: 3RGF). The 16 amino acids in the middle of the A-loop are not solved in the X-ray crystallography.

Figure 6. Hydrogen bond dynamics of human CDK8 A-loop during the 50-ns MD simulation

Hydrogen bond occupation of human CDK8 A-loop gradually increases in the first 20-ns MD simulation and stays relative stable during later 30-ns simulation.

Figure 7. Hydrogen bond analysis of the residues in human CDK8 A-loop during the 50-ns MD simulation

a, Average hydrogen bond distribution per residue (the residues were selected with >10% average hydrogen bond occupation) in human CDK8 A-loop; b, Hydrogen bond dynamics of K185 in human CDK8; c, Hydrogen bond dynamics of Leu179 in human CDK8 A-loop; d, Hydrogen bond dynamics of T196 in human CDK8 A-loop.

Figure 8. The molecular dynamics of CDK8 D173A, D189N, T196A and T196D structures

a, The CDK8 D173A structure with the lowest potential energy during the 50-ns MD simulation; b, The CDK8 D189N structure with the lowest potential energy during the 50-ns MD simulation; c, The CDK8 D196A structure with the lowest potential energy during the 50-ns MD simulation; d, The CDK8 T196D structure with the lowest potential energy during the 50-ns MD simulation.

Figure 9. The theoretical structure of human CDK8-CycC complex with the lowest potential energy in solution during the molecular dynamic simulation

a, The theoretical structure of human CDK8-CycC complex with the lowest potential energy in the water box; b, Overlap of MD simulated CDK8-CycC complex with the crystal CDK8-CycC complex. CDK8: cyan represents crystal structure and structure presentation represents MD structure. CycC: cyan represents crystal structure and orange represents MD structure.