www.biocristalografia.df.ibilce.unesp.br/alessandra
Molecular Model of Cyclin-Dependent Kinase 8
Alessandra R. S. Lente1 ; Adriana C. M. Permigiani1 ; Fernanda Canduri1,2 ; Helen A. Arcuri1,2, Walter F. Jr. De Azevedo1,2
1. Departamento de Física -UNESP/SÃO JOSÉ DO RIO PRETO-SP/BRAZIL.
2. Programa de Pós-Graduação em Biofisíca Molecular - UNESP /SÃO JOSÉ DO RIO PRETO-SP/BRAZIL.
Email: [email protected]
Introduction
Cell cycle progression is tightly controlled by the activity of cyclin-dependent kinases (CDKs). CDKs are inactive as monomers, and activation requires binding to cyclins, a diverse family of proteins whose levels oscillate during cell cycle. Many negative regulatory proteins (CDK inhibitors, CKIs)
have been discovered. The central role of CDKs in cell cycle regulation makes them a promissing target for discovering small inhibiting molecules that can modify the degree of cell proliferation. Since deregulation of cyclins and/or alteration or absence of CKIs have been associated with many
cancers, there is strong interest in structural studies of CDKs. The three-dimensional structure of CDK2 provides a structural foundation for understanding the mechanisms of activation and inhibition of CDK2 and for the discovery of inhibitions. Cyclin-dependent kinase 8 (cdk8) regulates
transcription by phosphorylating RNA polymerase II and TFIIH. The three-dimensional structure of human CDK8 has not yet been determined, then the apoenzyme and the CDK8 in complex with ATP have been modelled using human CDK2 as template.
Methods
There are two main approaches to homology modeling: 1) Fragment-based comparative modeling and 2) Restrained-based modeling. For modeling of the apoenzyme and CDK8:ATP we used the second approach (Sali & Blundell, 1993). Model building of apoenzyme and CDK8:ATP was
carried out using the program MODELLER (Sali & Blundell, 1993). This program is an automated approach to comparative modeling by satisfaction of spatial restraints. The modeling procedure begins with an alignment of the sequence to be modeled (target) with related known threedimensional structures (templates) (Figure 1). This alignment is usually the input to the program. The output is a three-dimensional model for the target sequence containing all main-chain and side-chain non-hydrogen atoms.
The N-terminus and the C-terminus were removed from the CDK8 sequence. The CDK2 template is shorter (298 residues), compared with 464
residues of the CDK8.
A total of 1000 models were generated for apoenzyme and CDK8:ATP, and the final models were select based on stereochemical quality. The
optimization of the complex was carried out by the use of the variable target function method employing methods of conjugate gradients and
molecular dynamics with simulated annealing. All modeling process was performed on a Beowulf cluster, with 16 nodes (B16/AMD Athlon 2100+;
BioComp, São José do Rio Preto, SP, Brazil). The overall stereochemical quality of the final model was assessed by the program PROCHECK
(Laskowski, 1994). The cutoff for hydrogen bonds and salt bridges was 3.5 Å.
Results and Discussion
Analysis of the model
Ramachandran diagram φ-ψ plots were generated to verify the stereochemical quality. CDK8 apoenzyme model shown 86.1% of the residues lie in
the most favorable regions and the remaining 11.7% in the additional allowed regions (Figure 2a). CDK8:ATP shown 86.8% in most favorable
regions and 10.4% of the residues in the additional allowed regions (Figure 2b). The residues in the generously allowed regions and in the
disallowed regions are in contact with solvent.
Figure 1: Sequence alignment of human CDK2 and CDK8. There are 35% of identity between the sequences. The alignment was
performed with the program MULTALIN (Corpet, 1988).
The Figure 6 shows a schematic drawing of apoenzyme (6a) and the binary complex CDK8-ATP (6b). The ATP molecule was found in
the cleft between the two lobes. The core (the β-sheet and the helical bundle) of the CDK8 structure is very similar to that of the
CDK2.
(a)
(b)
Figure 2: Ramachandran Plot of the CDK8. (a) without ATP model; (b) with ATP model.
.
The model generated shows that the structure is bi-lobate, like others protein kinases. The electrostatic potential surface of the model of CDK8
complexed with ATP is shown in Figure 3. The analysis of the charge distribution of the binding pockets indicates the presence of some charge
complementarity between ligand and enzyme, nevertheless most of the binding pocket is hydrophobic. The structure of molecular model was
superposed with your template (Figure 4). The r.m.s.d. value observed in the superposition was 0.99Å2.
Figure 6: Ribbon diagram of the human
CDK8 (a) apoenzyme (b) in complex with
ATP generated by Molscript (Kraulis, 1991)
and Raster3D (Merritt et al., 1997).
(b)
(a)
Table 1: Intermolecular hydrogen bonds between CDK8 and ATP.
Figure 3: Electrostatic potential surface of the
CDK8:ATP, calculated with GRASP (Nicholls et al.,
1991), shown from -10kT (red) to +10kT (blue).
Uncharged regions are in white.
Table 2: Intermolecular hydrogen bonds between CDK2 and ATP.
Figure 4: Superposition of the molecular model
(pink line) with template structure (yellow line).
Interactions of ATP with CDK8
The specificity and affinity between enzyme and its ligand depend on directional hydrogen bonds and ionic interactions, as well as on shape
complementarity of the contact surfaces of both partners (Canduri et al., 2001). For the binary complex of CDK8:ATP was observed a total of
15 hydrogen bonds (Table 1), as well as CDK2 in complex with ATP (Table 2). Figure 5 shows the stereo view of the interdomain interface
region illustrating the binding pockets of the molecular model (pink line) with your template structure (yellow line).
Conclusions
The analysis of CDK8 structural data indicates that differences between the apoenzyme and CDK8-ATP complex provide an explanation
for the results of earlier binding studies with ATP analogues and a basis for future inhibitor design.
References
Canduri, F.; Teodoro, L. G. V. L.; Lorenzi, C.C.B.; Hial, V.; Gomes, R.A.S.; Ruggiero Neto, J. and De Azevedo Jr, W. F. Acta Crystallogr. D57 (2001) 1560.
Corpet, F. Nucl. Acids Res. 16(22) (1988) 10881-10890.
De Azevedo Jr., W. F.; Gaspar, R.T.; Canduri, F.; Camera Jr., J.C. Biochem Biophys. Res. Commun. 297 (2002) 1154.
De Azevedo Jr., W. F.; Canduri, F. and da Silveira, N.J.F. Biochem Biophys. Res. Commun. 293 (2002) 566.
Figure 5: Stereo view of the
active site region of CDK8 in
complex with ATP
superimposed with the active
site of CDK2.
Kraulis, P. J. App. Cryst. 24 (1991) 946.
Merritt, E. A. and Bacon, D. J. Methods in Enzimology 277 (1997) 505-524.
Nicholls, A.; Sharp, K. A. and Honig, B. Proteins 11 (1991) 281.
Sali, A. and Blundell, T. L. J. Mol. Biol. 234 (1993) 779.