title: Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding.
abstract: Cyclin-dependent kinase 12 (CDK12) promotes transcriptional elongation by phosphorylation of the RNA polymerase II C-terminal domain (CTD). Structure-function studies show that this activity is dependent on a C-terminal kinase extension, as well as the binding of cyclin K (CycK). To better define these interactions we determined the crystal structure of the human CDK12/CycK complex with and without the kinase extension in the presence of AMP-PNP. The structures revealed novel features for a CDK, including a large β4-β5 loop insertion that contributes to the N-lobe interaction with the cyclin. We also observed two different conformations of the C-terminal kinase extension that effectively open and close the ATP pocket. Most notably, bound AMP-PNP was only observed when trapped in the closed state. Truncation of this C-terminal structure also diminished AMP-PNP binding, as well as the catalytic activity of the CDK12/CycK complex. Further kinetic measurements showed that the full length CDK12/CycK complex was significantly more active than the two crystallised constructs suggesting a critical role for additional domains. Overall, these results demonstrate the intrinsic flexibility of the C-terminal extension in CDK12 and highlight its importance for both ATP binding and kinase activity.
authors.affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Dixon-Clarke
authors.affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Elkins
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada.
authors.forename: S-W Grace
authors.initials: SW
authors.lastname: Cheng
authors.affiliation: Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada.|Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6H 3N1, Canada.
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Morin
authors.affiliation: Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
authors.forename: Alex N
authors.initials: AN
authors.lastname: Bullock
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000737:chemistry*; D000818:Animals; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D018844:Cyclin-Dependent Kinases / Q000737:chemistry*; D016213:Cyclins / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D061987:Sf9 Cells; D012995:Solubility; D018411:Spodoptera
chemical_list: C113028:CCNK protein, human; D016213:Cyclins; D000266:Adenylyl Imidodiphosphate; C447339:CDK12 protein, human; D018844:Cyclin-Dependent Kinases