Iveta Bártová

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

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin‐dependent 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.

A detailed analysis is presented of the dynamics of human CDK5 in complexes with the protein activator p25 and the purine-like inhibitor roscovitine. These and other findings related to the activation of CDK5 are critically reviewed from a molecular perspective. In addition, the results obtained on the behavior of CDK5 are compared with data on CDK2 to assess the differences and similarities between the two kinases in terms of (i) roscovitine binding, (ii) regulatory subunit association, (iii) conformational changes in the T-loop following CDK/regulatory subunit complex formation, and (iv) specificity in CDK/regulatory subunit recognition. An energy decomposition analysis, used for these purposes, revealed why the binding of p25 alone is sufficient to stabilize the extended active T-loop conformation of CDK5, whereas the equivalent conformational change in CDK2 requires both the binding of cyclin A and phosphorylation of the Thr160 residue. The interaction energy of the CDK5 T-loop with p25 is about 26 kcal·mol-1 greater than that of the CDK2 T-loop with cyclin A. The binding pattern between CDK5 and p25 was compared with that of CDK2/cyclin A to find specific regions involved in CDK/regulatory subunit recognition. The analyses performed revealed that the αNT-helix of cyclin A interacts with the α6-α7 loop and the α7 helix of CDK2, but these regions do not interact in the CDK5/p25 complex. Further differences between the CDK5/p25 and CDK2/cyclin A systems studied are discussed with respect to their specific functionality.

The mechanism of inhibition of the cyclin-dependent kinase-2 as revealed by the molecular dynamics study on the complex CDK2 with the peptide substrate HHASPRK

Molecular dynamics (MD) simulations were used to explain structural details of cyclin‐dependent kinase‐2 (CDK2) inhibition by phosphorylation at T14 and/or Y15 located in the glycine‐rich loop (G‐loop). Ten‐nanosecond‐long simulations of fully active CDK2 in a complex with a short peptide (HHASPRK) substrate and of CDK2 inhibited by phosphorylation of T14 and/or Y15 were produced. The inhibitory phosphorylations at T14 and/or Y15 show namely an ATP misalignment and a G‐loop shift (∼5 Å) causing the opening of the substrate binding box. The biological functions of the G‐loop and GxGxxG motif evolutionary conservation in protein kinases are discussed. The position of the ATP γ‐phosphate relative to the phosphorylation site (S/T) of the peptide substrate in the active CDK2 is described and compared with inhibited forms of CDK2. The MD results clearly provide an explanation previously not known as to why a basic residue (R/K) is preferred at the P2 position in phosphorylated S/T peptide substrates.

Analysis of CDK2 active-site hydration: a method to design new inhibitors.

The interactions between the protein and the solvent were analyzed, and protein regions with a high density of water molecules, as well as structural water molecules, were determined by using molecular dynamics (MD) simulations. A number of water molecules that were in contact with the protein for the whole trajectory were determined. Their interaction energies and hydrogen bonds with protein residues were analyzed. Altogether, 39, 27, 49, and 32 water molecules bound to the protein were found for trajectories of the free CDK2, CDK2/ATP, CDK2/roscovitine, and CDK2/isopentenyladenine complexes, respectively. Positions of observed water molecules were compared with X‐ray crystallography data. Special attention was paid to water molecules in the active site of the enzyme, and especially to the deep pocket, where the N9 roscovitine side‐chain is buried. Exchange of active‐site water molecules with bulk water through the tunnel from the pocket was observed. In the CDK2/isopentenyladenine complex simulation, two water molecules that arrange interaction between the inhibitor and the enzyme via an H‐bond were observed. Two stable water molecules in the trajectory of the free CDK2 were found that occupy the same position as the nitrogens N3 and N9 of the isopentenyladenine or N1 and N6 nitrogens of the adenosine triphosphate (ATP). The positions of structural water molecules were compared with the positions of substrate polar groups and crystallographic water molecules found in the Brookhaven Protein Data Bank for various CDK2 complexes. It was concluded that tracing tightly bound water molecules may substantially help in designing new inhibitors. Proteins 2004.

Regulatory phosphorylation of cyclin-dependent kinase 2: insights from molecular dynamics simulations

AbstractThe structures of fully active cyclin-dependent kinase-2 (CDK2) complexed with ATP and peptide substrate, CDK2 after the catalytic reaction, and CDK2 inhibited by phosphorylation at Thr14/Tyr15 were studied using molecular dynamics (MD) simulations. The structural details of the CDK2 catalytic site and CDK2 substrate binding box were described. Comparison of MD simulations of inhibited complexes of CDK2 was used to help understand the role of inhibitory phosphorylation at Thr14/Tyr15. Phosphorylation at Thr14/Tyr15 causes ATP misalignment for the phosphate-group transfer, changes in the Mg2+ coordination sphere, and changes in the H-bond network formed by CDK2 catalytic residues (Asp127, Lys129, Asn132). The inhibitory phosphorylation causes the G-loop to shift from the ATP binding site, which leads to opening of the CDK2 substrate binding box, thus probably weakening substrate binding. All these effects explain the decrease in kinase activity observed after inhibitory phosphorylation at Thr14/Tyr15 in the G-loop. Interaction of the peptide substrate, and the phosphorylated peptide product, with CDK2 was also studied and compared. These results broaden hypotheses drawn from our previous MD studies as to why a basic residue (Arg/Lys) is preferred at the P+2 substrate position. FigureView of the substrate binding site of the fully active cyclin-dependent kinase-2 (CDK2) (pT160-CDK2/cyclin A/ATP). The pThr160 activation site is located in the T-loop (yellow secondary structure). The G-loop, which partly forms the ATP binding site, is shown in blue. The Thr14 and Tyr15 inhibitory phosphorylation sites located in the G-loop are shown in licorice representation