Molecular dynamics modeling of the substitution of serine for the conservative glycine in the G loop in the yeast cdc28-srm mutant using the crystalline lattice of human kinase CDK2

Two-nanosecond molecular dynamics modeling of the crystalline lattice of an active complex of kinase pT160-CDK2/cyclin A/ATP-Mg2+ substrate has been performed. The results of modeling indicated that the structures of the nonmutant CDK2 complex and mutant CDK2 complex, which involves the G 16S-CD K2 substitution corresponding to that of yeast, markedly differ, the differences in structural conformations being particularly well pronounced in those regions that play a key role in the functioning of kinase. Based on the results of computations, structural elements are considered that may affect the kinase activity and regulatory phosphorylation, and the binding of protein kinase to cyclins and substrates.     

Structure and regulation of the CDK5-p25(nck5a) complex

CDK5 plays an indispensable role in the central nervous system, and its deregulation is involved in neurodegeneration. We report the crystal structure of a complex between CDK5 and p25, a fragment of the p35 activator. Despite its partial structural similarity with the cyclins, p25 displays an unprecedented mechanism for the regulation of a cyclin-dependent kinase. p25 tethers the unphosphorylated T loop of CDK5 in the active conformation. Residue Ser159, equivalent to Thr160 on CDK2, contributes to the specificity of the CDK5-p35 interaction. Its substitution with threonine prevents p35 binding, while the presence of alanine affects neither binding nor kinase activity. Finally, we provide evidence that the CDK5-p25 complex employs a distinct mechanism from the phospho-CDK2-cyclin A complex to establish substrate specificity.     

Conformational Equilibrium of CDK/Cyclin Complexes by Molecular Dynamics with Excited Normal Modes

Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr¹⁶⁰ phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.     

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G2 arrest in RCS chondrocytes

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G1 phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G2 phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G1 arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. The inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G2 arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and ATM/ATR kinase are known to play essential roles in the G2 checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G2 arrest. Additionally our results indicate that the transient G2 arrest is induced by FGF in RCS cell through mechanisms that are independent of the G1 arrest, and that the G2 block is not strictly required for the sustained G1 arrest but may provide a pausing mechanism that allows the FGF response to be fully established.     

Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.     

Homology modeling,molecular dynamic simulation and docking studies of cyclin dependent kinase 1

In order to develop promising cyclin dependent kinase 1 inhibitors, homology modeling, docking and molecular dynamic simulation techniques were applied to get insight into the functional and structural properties of cyclin dependent kinase 1 (CDK1). Since there is no reported CDK1 crystal structural data, the three dimensional structure of CDK1 was constructed based on homology modeling. An extensive dynamic simulation was also performed on a Flavopiridol-CDK1 complex for probing the binding pattern of Flavopiridol in the active site of CDK1. The binding modes of other inhibitors to CDK1 were also proposed by molecular docking. The structural requirement for developing more potent CDK1 inhibitors was obtained by the above-mentioned molecular simulations and pharmacophore modeling.     

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.     

The cyclin D1-dependent kinase associates with the pre-replication complex and modulates RB.MCM7 binding

The capacity of the cyclin D-dependent kinase to promote G(1) progression through modulation of RB.E2F is well documented. We now demonstrate that the cyclin D1/CDK4 kinase binds to components of the MCM complex. MCM7 and MCM3 were identified as cyclin D1-binding proteins. Catalytically active cyclin D1/CDK4 complexes were incorporated into chromatin-bound protein complexes with the same kinetics as MCM7 and MCM3, where they associated specifically with MCM7. Although the cyclin D1-dependent kinase did not phosphorylate MCM7, active cyclin D1/CDK4, but not cyclin E/CDK2, did catalyze the dissociation of an RB.MCM7 complex. Finally, expression of an active D1/CDK4 kinase but not cyclin E/CDK2 promoted the removal of RB from chromatin-bound protein complexes. Our data suggest that D1/CDK4 complexes play a direct role in altering an inhibitory RB.MCM7 complex possibly allowing for setting of the origin in preparation for DNA replication.     

CDK11p58 protein kinase activity is associated with Bcl-2 down-regulation in pro-apoptosis pathway

CDK11^p58, a G2/M-specific protein kinase, has been shown to be associated with apoptosis in many cell lines, with largely unknown mechanisms. Our previous study proved that CDK11^p58-enhanced cycloheximide (CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells. Here we report for the first time that ectopic expression of CDK11^p58 down-regulates Bcl-2 expression and its Ser70, Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells. Overexpression of Bcl-2 counteracts the pro-apoptotic activity of CDK11^p58. Furthermore, we confirm that the kinase activity of CDK11^p58 is essential to the down-regulation of Bcl-2 as well as apoptosis. Taken together, these results demonstrate that CDK11^p58 down-regulates Bcl-2 in pro-apoptosis pathway depending on its kinase activity, which elicits survival signal in hepatocarcinoma cells.     

Break CDK2/Cyclin E1 Interface Allosterically with Small Peptides

Most inhibitors of Cyclin-dependent kinase 2 (CDK2) target its ATP-binding pocket. It is difficult, however, to use this pocket to design very specific inhibitors because this catalytic pocket is highly conserved in the protein family of CDKs. Here we report some short peptides targeting a noncatalytic pocket near the interface of the CDK2/Cyclin complex. Docking and molecular dynamics simulations were used to select the peptides, and detailed dynamical network analysis revealed that these peptides weaken the complex formation via allosteric interactions. Our experiments showed that upon binding to the noncatalytic pocket, these peptides break the CDK2/Cyclin complex partially and diminish its kinase activity in vitro. The binding affinity of these peptides measured by Surface Plasmon Resonance can reach as low as 0.5 µM.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

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

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 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. Figure View 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     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human astrocytoma cells by the pyrrolo-1,5-benzoxazepine,PBOX-21

The present study examines the molecular mechanisms by which a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-21, induces G1 arrest in 1321N1 cells. PBOX-21-induced G1 arrest is preceded by both a decrease in CDK2 kinase activity, which is critical for the G1/S transition, and a downregulation in cyclin D(3) protein expression levels, suggesting that these two events may be crucially involved in the mediation of the cell cycle arrest. The decrease in CDK2 activity may be due to an observed decrease in CDK2 protein levels following PBOX-21 treatment. Coinciding with the arrest is a reduction in the activity of CDK4, due to either the observed PBOX-21 induced downregulation in CDK4 expression, or a reduction in complex formation between cyclin D(3)-CDK4 leading to a decrease in the levels of active cyclin D(3)-CDK4 complexes with kinase activity. The level of CDK6 activity was also seen to be reduced following PBOX-21 treatment, also possibly due to a reduction in complex formation with cyclin D(3). However, this reduction in CDK6 kinase activity was not seen until after PBOX-21-induced G1 arrest has reached its maximum, and therefore may be viewed as a consequence of, and a method of maintaining the PBOX-21-induced arrest, rather than a cause. Also in parallel with the G1 arrest elicited by PBOX-21 is an upregulation in the universal CDK inhibitor, p21. Furthermore, the retinoblastoma protein (Rb), a substrate of CDK2 and CDK6, whose phosphorylation is necessary for cell cycle progression, becomes hypophosphorylated. These results indicate that PBOX-21 exerts its growth inhibitory effects through the modulation of the expression and activity of several key G1 regulatory proteins.     

Key role for cyclin-dependent kinases in the first and second meiotic divisions of rat spermatocytes

In all systems examined so far, the G2/M phase transition is controlled by the M-phase promoting factor (MPF), a complex of cdc2 (CDK1) and cyclin B1. Histone H1 kinase activity and MPF components are present in pachytene spermatocytes (PS). However, it has not been demonstrated yet that direct inhibition of MPF activity prevents the G2/M transition in these cells. When roscovitine, a potent inhibitor of CDK1, CDK2, and CDK5 activities, was added to cocultures of PS with Sertoli cells, the number of both secondary spermatocytes and round spermatids formed were lower than in control cultures, despite similar cell viability. This effect of roscovitine was reversible, did not involve the Sertoli cells, and was dependent on the concentration of the inhibitor. Roscovitine did not modify the amount of MPF in these germ cells but inhibited the CDK1- or CDK2-associated histone H1 kinase activity of PS. Hence a functional relationship between cyclin-dependent kinase activity and the spontaneous processing of the first meiotic division and, for the first time, of the second meiotic division of male germ cells is shown.     

Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study

The current paradigm of cyclin-dependent kinase (CDK) regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1) Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD) simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg²⁺ ion at site 1 (Mg₁ ²⁺). The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds) between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg₁ ²⁺ ion, coupled with the Mg₂ ²⁺ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg₁ ²⁺ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg₁ ²⁺ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg₁ ²⁺ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.     

Comparative density functional theory based study of the reactivity of Cu,Ag, and Au nanoparticles and of (111) surfaces toward CO oxidation and NO2 reduction

Development of multi-target drugs is becoming increasingly attractive in the repertoire of protein kinase inhibitors discovery. In this study, we carried out molecular docking, molecular dynamics simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations, principal component analysis (PCA), and dynamical cross-correlation matrices (DCCM) to dissect the molecular mechanism for the valmerin-19 acting as a dual inhibitor for glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5). Detailed MM-PBSA calculations revealed that the binding free energies of the valmerin-19 to GSK3β/CDK5 were calculated to be ?12.60?±?2.28 kcal mol^-1 and ?11.85?±?2.54 kcal mol^-1, respectively, indicating that valmerin-19 has the potential to act as a dual inhibitor of GSK3β/CDK5. The analyses of PCA and DCCM results unraveled that binding of the valmerin-19 reduced the conformational dynamics of GSK3β/CDK5 and the valmerin-19 bound to GSK3β/CDK5 might occur mostly through a conformational selection mechanism. This study may be helpful for the future design of novel and potent dual GSK3β/CDK5 inhibitors.     

Ectopic expression of p27Kip1 in oligodendrocyte progenitor cells results in cell-cycle growth arrest

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27^Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 431–440, 1998     

TGF-beta induced G(1) cell cycle arrest requires the activity of the proteasome pathway. Transforming growth factor

Transforming growth factor-beta (TGF-beta) induces a potent G(1)/S-phase cell cycle arrest of epithelial cells by inhibiting the activities of cyclin D- and cyclin E-associated kinase complexes. Downregulation of the kinase activities is mediated by induction of cyclin dependent kinase (CDK) inhibitor p15(Ink4b) which blocks CDK4 and CDK6 kinases and leads to binding of p27(Kip1) to CDK2-cyclin E complex. Levels of several of these factors are controlled by the ubiquitin-proteasome pathway. We demonstrate here that proteasomal inhibitors release the cells from TGF-beta imposed G(1)-phase arrest and instigate the entry of the cells into S-phase. Proteasomal inhibitors are shown to specifically increase the activity of the cyclin D-kinase complex by increasing the levels of p27(Kip1) and cyclin D and by maintaining CDK4/6 protein levels leading to phosphorylation of the retinoblastoma protein without increasing cyclin E-associated kinase activity. The results indicate caution in the potential therapeutic use of the proteasome inhibitors due to unscheduled initiation of DNA replication in the presence of a physiological growth inhibitor.     

Allosteric Regulation of Cyclin-B Binding by the Charge State of Catalytic Lysine in CDK1 Is Essential for Cell-Cycle Progression

Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression.