Cyclin-dependent kinase 2 (CDK2) is a member of the CDK family of Ser/Thr protein kinase that is involved in cell cycle regulation. Protein kinases are known to depend on the Mg2+ metal ion to exert their catalytic activity, i.e., the transfer of a phosphoryl group from ATP to a substrate protein. Although Mg2+ is found in the active site of all protein kinases elucidated so far, it remains unknown regarding the number of Mg2+ ions essential for catalysis. In CDK2, contrary to the previous report that the enzyme only uses a single Mg2+ ion, recent studies suggest that it may require an additional Mg2+ion, which may be transient or bound just before catalysis. In this study, we address the question of whether CDK2 requires the second Mg2+ ion for its catalytic activity. In addition, we investigate the possibility of binding of other mono- or divalent-metal ions in the active site of CDK2, which is relevant if the second Mg2+ observed in the X-ray structure is an artifact of high Mg2+ concentration used. Molecular dynamics (MD) simulations and alchemical free energy calculations are used to determine the selectivity and specificity of CDK2 Mg2+ binding sites. We find that the two metal-binding sites exhibit different selectivity for Mg2+ compared to Na+ and Ca2+ ions. Overall, the second metal binding site has a relaxed selectivity towards Mg2+ compared to the first metal binding site which is highly selective towards Mg2+. Still, the Mg2+ binding affinities of both binding sites are substantially higher than those for Na+ and Ca2+. Our study also provides insights into the catalytic mechanism of CDK2, in particular the role of the second metal ion in orienting the substrate protein with respect to ATP, and may be useful in drug design efforts towards CDK2.

Is a second Mg2+ required for the catalytic activity of CDK2 enzyme: A computational study

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