Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Structures and Dynamics of the Complete Protein Kinase Catalytic Cycle of CDK2/CyclinA
Like many protein kinases, CDK2 is known to be a rather flexible enzyme and conformational transitions and protein dynamics are believed to play important roles in both the catalytic mechanism and the regulation of catalytic activity. We have determined high-resolution crystal structures of multiple steps along the complete reaction cycle of CDK2, including a transition-state complex consisting of CDK2/CyclinA bound to ADP, a substrate peptide and MgF3-, a structural mimic for the gamma-phosphate of ATP in the transition-state. Compared to structures of active CDK2 bound to its substrates or its products, the catalytic subunit of the kinase in the transition-state adopts a more closed conformation of the active site and, for the first time, a second catalytic Mg ion is observed in the active site. Coupled with a strong [Mg] effect on in vitro kinase activity, this structure suggests that the transient binding of a second Mg ion is necessary to achieve maximum rate-enhancement of the chemical reaction and Mg concentration could represent an important regulator of CDK2 activity in vivo. Molecular dynamics simulations illustrate how the simultaneous binding of substrate peptide, ATP and two Mg2+ ions is able to stabilize the closed and also more rigid organization of the active site that functions to orient the phosphates, stabilize the buildup of negative charge, and shield the subsequently activated gamma-phosphate from solvent. Once the phosphoryl-transfer step is complete, the second Mg is released and the active site returns to the more open conformation to release the products.
Matthew A. Young
Department of Biological Chemistry and Bioinformatics Program
University of Michigan, Ann Arbor, 48109.