The 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 Mg(2+) 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.

• MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   chemie   metabolismus   MeSH
• fosforylace MeSH
• katalytická doména MeSH
• lidé MeSH
• sekundární struktura proteinů MeSH
• threonin chemie   metabolismus   MeSH
• tyrosin chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasa 2 MeSH
• threonin MeSH
• tyrosin MeSH

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 A) 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 gamma-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 P(2) position in phosphorylated S/T peptide substrates.

• MeSH
• adenosintrifosfát chemie   MeSH
• aminokyselinové motivy MeSH
• časové faktory MeSH
• cyklin-dependentní kinasa 2 MeSH
• fosfáty chemie   MeSH
• fosforylace MeSH
• hořčík chemie   MeSH
• inhibitory enzymů chemie   MeSH
• ionty MeSH
• kinasy CDC2-CDC28 antagonisté a inhibitory   chemie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• peptidy chemie   MeSH
• rentgenové záření MeSH
• software MeSH
• stereoizomerie MeSH
• terciární struktura proteinů MeSH
• threonin chemie   MeSH
• tyrosin chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• CDK2 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasa 2 MeSH
• fosfáty MeSH
• hořčík MeSH
• inhibitory enzymů MeSH
• ionty MeSH
• kinasy CDC2-CDC28 MeSH
• peptidy MeSH
• threonin MeSH
• tyrosin MeSH

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.

• MeSH
• aktivace enzymů MeSH
• cyklin-dependentní kinasa 2 MeSH
• fosforylace MeSH
• fosfotyrosin metabolismus   MeSH
• glycin metabolismus   MeSH
• kinasy CDC2-CDC28 antagonisté a inhibitory   chemie   metabolismus   MeSH
• molekulární modely MeSH
• sekundární struktura proteinů MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasa 2 MeSH
• fosfotyrosin MeSH
• glycin MeSH
• kinasy CDC2-CDC28 MeSH