Concestor kinase activation mechanism uncovers the cyclin dependence of CDK family kinases.
Evolution has altered the free energy landscapes of protein kinases to introduce different regulatory switches and alters their catalytic functions. An understanding of evolutionary pathways behind these changes at atomistic resolution is of great importance for drug design. In this work, we demonstrate how cyclin dependency has emerged in cyclin-dependent kinases (CDKs) by reconstructing their closest experimentally characterized cyclin-independent ancestor. Using available crystal structures of CDK2, regulatory switches are identified and four possible hypotheses describing why CDK2 requires an extra intra-domain regulatory switch compared to the ancestor are formulated. Each hypothesis is tested using all-atom molecular dynamics simulations. Both systems show similar stability in the K33-E51 hydrogen bond and in the alignment of residues in the regulatory-spine, two key protein kinase regulatory elements, while auto-inhibition due to a helical turn in the a-loop is less favorable in the ancestor. The aspartate of the DFG motif does not form a bidentate bond with Mg in CDK2, unlike the ancestor. Using the results of hypothesizes testing, a set of mutations responsible for the changes in CDK2 are identified. Our findings provide a mechanistic rationale for how evolution has added a new regulatory switch to CDK proteins. Moreover, our approach is directly applicable to other proteins.