Inclining the Purine Base Binding Plane in Protein Kinase CK2 by Exchanging the Flanking Side-chains Generates a Preference for ATP as a Cosubstrate Original Research Article

Protein kinase CK2 (casein kinase 2) is a highly conserved and ubiquitously found eukaryotic serine/threonine kinase that plays a role in various cellular key processes like proliferation, apoptosis and circadian rhythm. One of its prominent biochemical properties is its ability to use GTP as well as ATP as a cosubstrate (dual-cosubstrate specificity). This feature is exceptional among eukaryotic protein kinases, and its biological significance is unknown. We describe here a mutant of the catalytic subunit of protein kinase CK2 (CK2α) from Homo sapiens (hsCK2α) with a clear and CK2-atypical preference for ATP compared to GTP. This mutant was designed on the basis of several structures of CK2α from Zea mays (zmCK2α) in complex with various ATP-competitive ligands. A structural overlay revealed the existence of a “purine base binding plane” harbouring the planar moiety of the respective ligand like the purine base of ATP and GTP. This purine base binding plane is sandwiched between the side-chains of Ile66 (Val66 in hsCK2α) and Met163, and it adopts a significantly different orientation than in prominent homologues like cAMP-dependent protein kinase (CAPK). By exchanging these two flanking amino acids (Val66Ala, Met163Leu) in hsCK2α1–335, a C-terminally truncated variant of hsCK2α, the cosubstrate specificity shifted in the expected direction so that the mutant strongly favours ATP. A structure determination of the mutant in complex with an ATP-analogue confirmed the predicted change of the purine base binding plane orientation. An unexpected but in retrospect plausible consequence of the mutagenesis was, that the helix αD region, which is in the direct neighbourhood of the ATP-binding site, has adopted a conformation that is more similar to CAPK and less favourable for binding of GTP. These findings demonstrate that CK2α possesses sophisticated structural adaptations in favour of dual-cosubstrate specificity, suggesting that this property could be of biological significance.