Identifying allosteric fluctuation transitions between different protein conformational states as applied to Cyclin Dependent Kinase 2

BACKGROUND: The mechanisms underlying protein function and associated conformational change are dominated by a series of local entropy fluctuations affecting the global structure yet are mediated by only a few key residues. Transitional Dynamic Analysis (TDA) is a new method to detect these changes in local protein flexibility between different conformations arising from, for example, ligand binding. Additionally, Positional Impact Vertex for Entropy Transfer (PIVET) uses TDA to identify important residue contact changes that have a large impact on global fluctuation. We demonstrate the utility of these methods for Cyclin-dependent kinase 2 (CDK2), a system with crystal structures of this protein in multiple functionally relevant conformations and experimental data revealing the importance of local fluctuation changes for protein function. RESULTS: TDA and PIVET successfully identified select residues that are responsible for conformation specific regional fluctuation in the activation cycle of Cyclin Dependent Kinase 2 (CDK2). The detected local changes in protein flexibility have been experimentally confirmed to be essential for the regulation and function of the kinase. The methodologies also highlighted possible errors in previous molecular dynamic simulations that need to be resolved in order to understand this key player in cell cycle regulation. Finally, the use of entropy compensation as a possible allosteric mechanism for protein function is reported for CDK2. CONCLUSION: The methodologies embodied in TDA and PIVET provide a quick approach to identify local fluctuation change important for protein function and residue contacts that contributes to these changes. Further, these approaches can be used to check for possible errors in protein dynamic simulations and have the potential to facilitate a better understanding of the contribution of entropy to protein allostery and function

High-resolution crystal structures of human cyclin-dependent kinase 2 with and without ATP: Bound waters and natural ligand as guides for inhibitor design

Inhibition of the cell cycle is widely considered as a new approach toward treatment for diseases caused by unregulated cell proliferation, including cancer. Since cyclin-dependent kinases (CDKs) are key enzymes of cell cycle control, they are promissing targets for the design and discovery of drugs with antiproliferative activity. The detailed structural analysis of CDK2 can provide valuable information for the design of new ligands that can bind in the ATP binding pocket and inhibit CDK2 activity. For this objective, the crystal structures of human CDK2 apoenzyme and its ATP complex were refined to 1.8 and 1.9 Angstrom, respectively. The high-resolution refinement reveals 12 ordered water molecules in the ATP binding pocket of the apoenzyme and five ordered waters in that of the ATP complex. Despite a large number of hydrogen bonds between ATP-phosphates and CDK2, binding studies of cyclic AMP-dependent protein kinase with ATP analogues show that the triphosphate moiety contributes little and the adenine ring is most important for binding affinity. Our analysis of CDK2 structural data, hydration of residues in the binding pocket of the apoenzyme, flexibility of the ligand, and structural differences between the apoenzyme and CDK2-ATP complex provide an explanation for the results of earlier binding studies with ATP analogues and a basis for future inhibitor design