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Structural Analysis of the Inhibition of Cdk4 and Cdk6 by p16

INK4a

throughMolecularDynamics Simulations

http://www.jbsdonline.com

Abstract

Cyclin-dependent kinases 4, 6 and 2 (Cdk4/6/2), are proteins that lead progression throughthe G1-S transition, a step strictly regulated in the process of cell proliferation. The p16

INK4a

tumor suppressor, whose expression is inhibited in a high number of cancers, binds toCdk4/6 and inhibits phosphorylation of the retinoblastoma protein, forcing cells to remain inthe G1 phase and therefore, arresting cell division. Accordingly, the design of small com-pounds mimicking the inhibition of p16

INK4a

appears to be a promising way to treat cancer.In order to get some insight into the key interactions governing recognition between differ-ent cyclin-dependent kinases and the p16

INK4a

tumor suppressor, the present work reportsthe results of molecular dynamics simulations of both, the Cdk6-p16

INK4a

complex and theCdk4-p16

INK4a

complex, respectively at 300 K. Most of the key interactions observed, werealready anticipated in the analysis of the crystal structure of Cdk6-p16

INK4a

. However, a fewdifferent features found out from the analysis of these calculations provide a better under-standing of the role of the T-loop conformation, a fragment of Cdks, and the way the ATPbinding-site is distorted upon binding of p16

INK4a

.

Introduction

Progression through the different phases of the cell cycle is controlled at severalcheckpoints, where continuation or arrest of the process is decided (1).Specifically, checkpoint at the G1-S transition is crucial, since there is no furtherextra cellular control of cell duplication (2). Thus, after this checkpoint cell divi-sion proceeds irreversibly unless a fatal misduplication of DNAis detected, inwhich case apoptosis pathways are activated (3). The G1 to S phase transition iscontrolled by the cyclin dependent kinases Cdk2, Cdk4 and Cdk6, being the twolatter directly implicated in process carried out at the G1-S checkpoint (2). Thisprocess begins with the activation of the Cdk4/6 through external proliferative sig-nals that provide them with kinase activity. In a second step, these proteins phos-phorilate the retinoblastoma protein (pRb) with the subsequent release of the tran-scriptional factor E2F, which in turn activates the synthesis of necessary proteinsfor the S phase (2). Checkpoint dysregulation induces cells to enter in a non-con-trolled proliferative cycle, being the cause of different cancers.Cdk4/6 are activated upon binding to cyclin D, as well as by phosphorylation atresidues Thr

177

in Cdk6, or Thr

172

in Cdk4, by a Cdk activating kinase (Cak).Before promoting the synthesis of the different players for the next phase, severalstimuli exert checkpoint control to inactivate downstream transcription. Thisprocess can be performed by dephosphorylation of the Cdks by phosphatases,although it can also be accomplished by either kinases that phosphorilate Cdks atspecific threonine or serine residues or by inhibitors that bind to the Cdks prevent-

Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102Volume 20, Issue Number 3, (2002)©Adenine Press (2002)

ÓscarVillacañas

1

Juan J. Pérez

2

Jaime Rubio-Martínez

1*

1

Department de Química FísicaUniversitat de BarcelonaMartí i Franquès, 108028 Barcelona, Spain

2

Departament d'Enginyeria Química(UPC)ETS d'Enginyeria IndustrialAv. Diagonal, 64708028 Barcelona, Spain

347

*

Phone: 0034-93-4021222Fax: 0034-93-4021231Email: j.rubio@qf.ub.es

ing them from acquiring their active conformation (4, 5). Specifically, Ink4 is afamily of inhibitors that have Cdk4 and Cdk6 as target proteins. There are fourmembers of this family of inhibitors described so far, all exhibiting in common sev-eral ankyrin-like repeats: p16

INK4a

(henceforth p16), p15

INK4b

(henceforth p15),p18

INK4c

(henceforth p18) and p19

INK4d

(henceforth p19) (5).Interestingly, p16 has been found mutated or non-expressed more often than theother members of the Ink4 family in numerous types of cancer (1), pointing to thepivotal role that this protein plays in the process of cell cycle progression at the G1-S checkpoint. Accordingly, a better understanding of the key interactions betweenp16 and Cdk4/6 is necessary in order to gain information relevant for designingsmall molecule mimics of p16. This process can be considered as a first step in thedevelopment of new therapeutic agents to treat cancer.After analysis of the diverse experimental information gathered from the literature(6, 7) and from the analysis of the available crystal structures of different Cdk6-Ink4 complexes, such as Cdk6-p16 (8), Cdk6-p19 (8, 9) and cyclinK-Cdk6-p18(10), it can be shown that Ink4 inhibitors can bind either to the free Cdk4/6 or tothe CyclinD-Cdk4/6 complex, exhibiting very similar contacts (10). Accordingly,the Cdk6-p16 complex can be considered as a prototype to study the structuralbasis of the tumor suppressor p16 inhibition.Although the analysis of the crystal structure of the Cdk6-p16 complex solved byX-ray diffraction methods reveals most of the key interactions involved in the inter-action between these two proteins, it provides a static picture of the complex thatrequires further investigation. Molecular dynamics simulations can provide com-plementary information on the nature of these interactions, since the experimentalconditions necessary to obtain single crystals may differ from those of the activecomplex. Accordingly, the present work presents the results of molecular dynam-ics simulations of Cdk6-p16 and Cdk4-p16 complexes at 300 K. The trajectorieswere analyzed to confirm, discard or discover new interactions not accounted fromthe analysis of the crystal structures.

Methods

All the calculations described in the present work were carried out at the molecularmechanics level using the parm94 force field (11) with the AMBER suite of programs(12).

In vacuo

calculations were carried out with a dielectric constant of 4r and a

cut-off

of 11Å to compute non-bonded interactions. In those calculations where the sol-vent was considered explicitly (TIP3Pwater), the dielectric constant was changed to1r, and the

cutoff

was kept to 11Å to compute the non bonded interactions (13).

Construction of the Cdk6-p16 Complex

The 3D structure of the complex Cdk6-p16 available from the Protein Data Bank(entry 1BI7) comprises residues 10-48 and 72-301 of Cdk6, and residues 10-135 of p16. The coordinates of the missing residues could not be resolved from the X-raydiffraction studies due to their flexibility (8). Although the missing residues of bothtermini are presumably not relevant for the function of the complex, the fragment 49-71 of Cdk6 includes the so-called PLSTIRE helix, known to be implicated in Cdksactivity (14). Accordingly, the coordinates of this fragment were constructed byhomology modeling using the 3D structure of the complex Cdk6 (human)-p19(mouse) as template (9). In order to remove the most important steric stress, the com-plex was subject to energy minimization

in vacuo

using an effective dielectric constantof 4r. After this process, a cap of TIP3Pwater molecules, centered at the Cdk6-p16center of masses and with a radius of 41 Å was added to the complex. The complexwas again energy minimized following a three-step procedure using the belly option.First, water molecules were allowed to relax while the atoms of the protein were kept

348Villacañas et al.

frozen. In a second step, only side chains of the residues as well as water moleculeswere relaxed and finally, in the third step the all the atoms were allowed to move.

Construction of the Cdk4-p16 Complex

The Cdk4-p16 complex was constructed by homology modeling following the align-ment of Brotherton (9) and using the 3D structure of the previous Cdk6-p16 complexas template. For this purpose, the structures of Cdk4 and Cdk6 were assumed toexhibit the same fold, since the two proteins share a 70% of sequence identity (15).Geometry optimization of the Cdk4-p16 complex was carried out in vacuo followinga three step procedure, and taking into account the different structural manipulationsundertaken during the homology modeling process. First, the N-terminal fragmentincluding all residues up to Leu

34

of Cdk4 was energy minimized due to the deletionof residue Leu

33

of the template protein in the constructed model. Second, the N-ter-minal fragment up to Ser

52

of Cdk4 was optimized due to the insertion of threeglycines at positions 42-44 in Cdk4, which are not present in the template. Finally,since side chain coordinates of non-identical residues were generated using the EDITmodule of AMBER 4.1 (12), all side chains were allowed to move. In a further step,the complex was solvated with a cap of TIP3Pwater molecules centered at center of masses of the complex with a radius of 41Å, and minimized following the same pro-cedure as followed to refine the Cdk6-p16 complex. The quality of the structuremodeled was assessed using the program PROCHECK (16), which reported that 96%of the residues fell within the allowed regions of the Ramachandran plot.

Molecular Dynamics Simulations

Molecular dynamics (MD) simulations of both complexes were performed at a con-stant temperature of 300K by coupling the system to a thermal bath, usingBerendsen’s algorithm as implemented in the AMBER program (17), with a timecoupling constant of 0.2 ps. Time step was set to 1fs, and the list of nearest neigh-bors was updated every 15 steps. In order to reduce the CPU time of the computa-tion, the radius of the sphere of water molecules was reduced to a 31Å that allow-ing the inclusion of the p16 inhibitor and those Cdk residues that are closer than 12Åto the tumor suppressor. Accordingly, the contact surface between the two proteinswas properly solvated. In these conditions, all residues of Cdk located at longer dis-tances of 12Å of p16 were frozen during the MD calculations using the belly option.MD trajectory calculations began by heating the minimized structure up to 300Kover a period of 100ps at a constant rate of 30K/10ps, followed by a 1ns dynamicscalculation at a constant temperature of 300K.All the figures of the present work were carried out with VMD graphics program (18).

Results and Discussion

Figure 1 shows the fluctuation of the total energy along the 1 ns trajectories com-puted for both complexes at 300K. Whereas the Cdk6-p16 complex exhibited aconstant energy few picoseconds after the heating process, the Cdk4-p16 complexcould only be equilibrated the last 600 ps of simulation. This must be due to thedifferent origin of the starting structures. Thus, since the Cdk4-p16 complex wasmodeled by homology modeling, it is possible that some side chains were not prop-erly accommodated and a longer equilibration time was required. Accordingly,only the last 600 ps of both complexes were considered as production and used forthe subsequent structural analysis.Analysis of the rmsd (root mean square deviation) values of both proteins, p16 andCdk6, in regard to the average structure of the complex during the last 600 ps(Figures 2a), show that the former exhibits a higher oscillatory behavior, suggest-

349Molecular Dynamics Study ofInhibitors of Cdk4/6

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