Catalytic activity of mutants of yeast protein kinase CK2α

Yeast CK2 is a highly conserved member of the protein kinase CGMC subfamily composed of two catalytic (α and α') and two regulatory (β and β') subunits. The amino-acid sequences of both catalytic subunits are only 60% homologous. Modelling of the tertiary structure of the CK2α displays additional α-helical structures not present in the CK2α' subunit, connecting the ATP-binding loop with the catalytic and activation loops. Deletion of this part causes drastic structural and enzymatic changes of the protein (CK2αΔ91-128) with characteristics similar to yeast CK2α' (low sensitivity to salt, heparin and spermine). Additionally, the deletion causes an over 5-fold decrease of the binding affinity for ATP and ATP-competitive inhibitors (TBBt and TBBz). The structural basis for TBBt and TBBz selectivity is provided by the hydrophobic pocket adjacent to the ATP/GTP binding site, which is smaller in CK2 than in the majority of other protein kinases. The importance of hydrophobic interactions in the binding of specific inhibitors was investigated here by mutational analysis of CK2α residues whose side chains contribute to reducing the size of the hydrophobic pocket. Site-directed mutagenesis was used to replace Val67 and Ile213 by Ala. The kinetic properties of the single mutants CK2αVal67Ala and CK2αIle213Ala, and the double mutant CK2Val67Ala Ile213Ala were studied with respect to ATP, and both inhibitors TBBt and TBBz. The Km values for ATP did not change or were very close to those of the parental kinase. In contrast, all CK2α mutants analysed displayed higher Ki values towards the inhibitors (10 to 12-fold higher with TBBt and 3 to 6-fold with TBBt) comparing to recombinant wild-type CK2α.