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.

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). Tennanosecond-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 ␥-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.

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 Thr 160 residue. The interaction energy of the CDK5 T-loop with p25 is about 26 kcal⅐mol ؊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 ␣NT-helix of cyclin A interacts with the ␣6-␣7 loop and the ␣7 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.Cyclin-dependent kinases (CDKs) 3 control the progression of the cell cycle (1) and participate in a subset of apoptosis programs (1-3). CDKs consist of two subunits, one catalytic and the other regulatory. The catalytic subunit is a Ser/Thr kinase from the CMGC kinase family (4); the associated regulatory proteins are called cyclins (1). Cyclins are highly specific for individual kinases, resulting in the formation of distinct complexes, e.g. CDK1/cyclin B1 and CDK2/cyclin E1. The primary biological function of these complexes is related to regulation of the cell cycle. However, certain CDKs do not participate in cell cycle control, instead they are involved in controlling cell differentiation in neuronal and muscle cells. This class of CDKs is exemplified by CDK5, which plays a critical role during neuronal development (5-8).Several CDKs (CDK1-4 and CDK6) show a dual mechanism of activation based on the binding of the cyclin box fold (CBF) region of the regulatory subunit to the kinase and phosphorylation of the activation loop (also known as the T-loop) of the kinase (1). However, this mode of activation is not observed in CDK5, despite sequence identities of almost 60% for CDK2-CDK5 pairs in different species (Scheme 1). CDK5 is a unique member of the CDK family, because it is not activated by a cyclin. It binds to cyclins D and E, but they fail to induce its kinase activity (5, 6). Instead, CDK5 activity is triggered by p35 NCKA and p39 NCKAI (henceforth referred to as p35 and p39, respec...

Cyclin-dependent kinase 2 (CDK2) is the most thoroughly studied of the cyclin-dependent kinases that regulate essential cellular processes, including the cell cycle, and it has become a model for studies of regulatory mechanisms at the molecular level. This contribution identifies flexible and rigid regions of CDK2 based on temperature B-factors acquired from both X-ray data and molecular dynamics simulations. In addition, the biological relevance of the identified flexible regions and their motions is explored using information from the essential dynamics analysis related to conformational changes of CDK2 and knowledge of its biological function(s). The conserved regions of CMGC protein kinases' primary sequences are located in the most rigid regions identified in our analyses, with the sole exception of the absolutely conserved G13 in the tip of the glycine-rich loop. The conserved rigid regions are important for nucleotide binding, catalysis, and substrate recognition. In contrast, the most flexible regions correlate with those where large conformational changes occur during CDK2 regulation processes. The rigid regions flank and form a rigid skeleton for the flexible regions, which appear to provide the plasticity required for CDK2 regulation. Unlike the rigid regions (which as mentioned are highly conserved) no evidence of evolutionary conservation was found for the flexible regions.