Activation and inhibition of cyclin‐dependent kinase‐2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine‐rich loop

Abstract

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclindependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/ Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.

Different Mechanisms of CDK5 and CDK2 Activation as Revealed by CDK5/p25 and CDK2/Cyclin A Dynamics

“…By contrast, the mechanism of inhibition by Thr 14 phosphorylation may be similar to the mechanism of CDK2 inhibition by Thr 14 phosphorylation, which results in significant misalignment of ATP in the active site because of electrostatic repulsion between two adjacent negatively charged groups, i.e. the phosphate moiety of ATP and that of Thr(P) 14 (19,20). Consequently, the misaligned ATP ␥-phosphate cannot be transferred to the substrate Ser/Thr.…”

“…All analyses of the MD simulations were carried out using the CARNAL, ANAL, and PTRAJ modules of AMBER-6.0 (32), and with GROMACS (33). The method of Cornell et al (34) was used for the parameterization of roscovitine and the phosphorylated tyrosine (19) and threonine residues. The results obtained were compared with previously obtained data on the fully active form of CDK2 (Thr(P) 160 -CDK2/cyclin A/ATP) and the fully active form of CDK2 complexed with the peptide substrate HHASPRK (Thr(P) 160 -CDK2/cyclin A/ATP/HHASPRK) (19,20).…”

“…The method of Cornell et al (34) was used for the parameterization of roscovitine and the phosphorylated tyrosine (19) and threonine residues. The results obtained were compared with previously obtained data on the fully active form of CDK2 (Thr(P) 160 -CDK2/cyclin A/ATP) and the fully active form of CDK2 complexed with the peptide substrate HHASPRK (Thr(P) 160 -CDK2/cyclin A/ATP/HHASPRK) (19,20). The results of an MD simulation of the CDK2/roscovitine complex (25) were used to compare the interactions of roscovitine with CDK5 to those with CDK2.…”

“…The phosphorylation of Thr 14 and Tyr 15 in the glycine-rich loop (G-loop; residues 11-16) that forms the "ceiling" of the ATP-binding site is important in the regulation of CDK activity (18). Certain structural aspects of CDK2 were recently proposed on the basis of molecular dynamics simulations (19,20). Inhibitory phosphorylation of specific residues (Thr 14 and Tyr 15 ) in CDK2 results in ATP misalignment and a shift of ϳ5 Å in the position of the G-loop, "opening" the substratebinding site.…”

Variability of the Cyclin-Dependent Kinase 2 Flexibility Without Significant Change in the Initial Conformation of the Protein or Its Environment; a Computational Study

“…There are several reports that have studied the flexibility of the hCDK2 protein, applying a comparative approach (18)(19)(20)(21)(22), that is studying the flexibility of this protein in significantly different conditions of the protein structure or environment, such as phosphorylation, ATP binding, protein binding etc. However, in our work we have studied variations and changes in the flexibility of hCDK2 protein with no significant changes in its initial environment, structure or even its conformation.…”