biostudies-literatureCushing VICancer Research UKMedical Research CouncilWellcome Trust911-917https://www.ebi.ac.uk/biostudies/studies/S-EPMC7618291biostudies-literatureUnknown390(6776)Cyclin-dependent kinases (CDKs) are prototypical regulators of the cell cycle. The CDK-activating kinase (CAK) acts as a master regulator of CDK activity by catalyzing the activating phosphorylation of CDKs on a conserved threonine residue within the regulatory T-loop. However, structural data illuminating the mechanism by which the CAK recognizes and activates CDKs have remained elusive. In this study, we determined high-resolution structures of the CAK in complex with CDK2 and CDK2-cyclin A2 by cryogenic electron microscopy. Our structures reveal a T-loop-independent kinase-kinase interface with contributions from both kinase lobes. Computational analysis and structures of the CAK in complex with CDK1-cyclin B1 and CDK11 indicate that these structures represent the general architecture oScience (New York, N.Y.)Structural basis of T-loop-independent recognition and activation of CDKs by the CDK-activating kinase.PMC761829128159MR/V009354/1Cushing VIDan LMMcGeoch AJSFeng JDavey NEWilliams SLChoudhary JSGreber BJRoumeliotis TIfalseStructural basis of T-loop-independent recognition and activation of CDKs by the CDK-activating kinase.Cyclin-dependent kinases (CDKs) are prototypical regulators of the cell cycle. The CDK-activating kinase (CAK) acts as a master regulator of CDK activity by catalyzing the activating phosphorylation of CDKs on a conserved threonine residue within the regulatory T-loop. However, structural data illuminating the mechanism by which the CAK recognizes and activates CDKs have remained elusive. In this study, we determined high-resolution structures of the CAK in complex with CDK2 and CDK2-cyclin A2 by cryogenic electron microscopy. Our structures reveal a T-loop-independent kinase-kinase interface with contributions from both kinase lobes. Computational analysis and structures of the CAK in complex with CDK1-cyclin B1 and CDK11 indicate that these structures represent the general architecture o2025-01-01T00:00:00Z2025 Nov2026-06-11T05:01:57.379Z2026-06-11T03:08:15.758ZS-EPMC76182914110058510.1126/science.adw0053