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.
Highlights
Cyclin-dependent kinases (CDK1-20) form a large family of serine/threonine protein kinases that depend on the binding of specific cyclin proteins for maximal catalytic activity[1,2]
Cyclin-dependent kinase 12 (CDK12) complexes with cyclin L1 or L2 have been reported to localise to nuclear speckles and to regulate RNA splicing[12,27]
We compared the kinetic parameters of our proteins and show that the full length CDK12/cyclin K (CycK) complex remains significantly more active suggesting additional structural elements are required for optimal function
Summary
Cyclin-dependent kinases (CDK1-20) form a large family of serine/threonine protein kinases that depend on the binding of specific cyclin proteins for maximal catalytic activity[1,2]. Many CDKs require phosphorylation by the CDK activating kinase (CAK; comprising CDK7/Cyclin H/MAT1 in humans), which targets a conserved threonine residue within the kinase activation loop[10,11]. This both stabilizes the active configuration of the CDK/cyclin complex and establishes greater interaction with the substrate[7]. We report further structures of CDK12/CycK complexes solved in the presence of AMP-PNP and containing variable truncations of the C-terminal kinase extension. We compared the kinetic parameters of our proteins and show that the full length CDK12/CycK complex remains significantly more active suggesting additional structural elements are required for optimal function
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