Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding

Sci Rep. 2015 Nov 24:5:17122. doi: 10.1038/srep17122.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Imidodiphosphate / chemistry*
  • Animals
  • Catalytic Domain
  • Crystallography, X-Ray
  • Cyclin-Dependent Kinases / chemistry*
  • Cyclins / chemistry*
  • Humans
  • Hydrogen Bonding
  • Models, Molecular
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Structure, Quaternary
  • Sf9 Cells
  • Solubility
  • Spodoptera

Substances

  • CCNK protein, human
  • Cyclins
  • Adenylyl Imidodiphosphate
  • CDK12 protein, human
  • Cyclin-Dependent Kinases

Associated data

  • PDB/4CXA
  • PDB/4UN0