Protein-protein interactions studies can greatly increase the amount of structural and functional information pertaining to biologically active molecules and processes. The information obtained from such studies can lead to design and application of new modification in order to obtain a desired bioactivity. Many application packages and servers performing docking, such as HEX, DOT, AUTODOCK, and ZDOCK are now available for predicting the lowest free energy state of a protein complex. In this study, we have focused on cyclin-dependent kinase 4 (Cdk4), a key molecule in the regulation of cell cycle progression at the G1-S phase restriction point and p16INK4a, a tumor suppressor which inhibits Cdk4 activity. Truncated structures were created to find the more critical regions of p16 for interaction. The tertiary structures were determined by ProSAL, GENO3D Web Server. We evaluated their interactions with Cdk4 using two docking systems, HEX 4.5 and DOT 1. Calculations were performed on a high-speed computer. Minimizations and visualizations were carried out by PdbViewer 3.7. Considering shape and shape/electrostatic total energy, structures containing ANK II, III and IV motifs that lack the N-terminal region of the full length p16 molecule showed the best fit complexes among the p16 truncated forms. The free energies were compatible with that of p16 full length original form, the full length. It seems that the N-terminal of the molecule is not crucial for the interaction since the truncated structure containing only this region did not show a good total energy.
The tumor suppressor p16INK4a has earned widespread attention in cancer studies since its discovery as an inhibitor of cyclin-dependent kinases (CDKs) 4/6. Structurally, it consists of four complete ankyrin repeats, believed to be involved in CDK4 interaction. According to the previous disparities concerning the importance of domains and inactivating mutations in p16, we aimed to search for the domain possessing the functional properties of the full length protein. Upon our in silico screening analyses followed by experimental assessments, we have identified the novel minimum functional domain of p16 to be the C-terminal half including ankyrin repeats III, IV and the C-terminal flanking region accompanied by loops 2 and 3. Transfection of this truncated form into HT-1080 human fibrosarcoma cells, lacking endogenous p16, revealed that it is able to inhibit cell growth and proliferation equivalent to p16 INK4a . The functional analysis showed that this fragment like p16 can interact with CDK4/6, block the entry into S phase of the cell cycle and suppress growth as indicated by colony formation assay. Identification of p16 minimum functional domain can be of benefit to the future peptidomimetic drug design as well as gene transfer for cancer therapy.
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