The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

Abstract

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.

Figure 1.

Calorimetric and structural analysis of the CK2α/CK2β interaction. (A) DSC curves of hsCK2α^1–335 (red), hsCK2β^1–193 (blue), and the corresponding holoenzyme [(hsCK2α^1–335)₂(hsCK2β^1–193)₂] (black). One representative example out of three repetitions, respectively, is drawn; the indicated melting points are the corresponding average values. (B) ITC profile of the hsCK2α^1–335/hsCK2β^1–193 interaction. A representative example out of three ITC runs is documented. The upper half shows the original heat production upon injection and the lower one the integrated and dilution corrected peaks. The final thermodynamic parameters in the inset are average values over three repetitions. (C) The CK2β binding region of human CK2α. The figure is based on the structure of hsCK2α^1–335 in complex with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) (2RKP) (Raaf et al. 2008). After structural superimposition, the β4β5-loops of hsCK2α^1–335 in complex with sulfate ions (1PVR) (Niefind et al. 2007) (black color) and of a hsCK2α chain within the CK2 holoenzyme (1JWH) (Niefind et al. 2001) (blue color) are drawn. Some hydrophobic side chains of the interface (yellow) and of the tip of the β4β5-loop (gray: closed conformation; blue: open conformation) are added. The figure was prepared with BRAGI (Schomburg and Reichelt 1988).

Figure 2.

Structure of hsCK2β^1–193. (A) Overall ribbon presentation of the hsCK2β^1–193 dimer. Each hsCK2β^1–193 monomer consists of a body comprising two domains (subunit A: blue/red; subunit B: green/yellow) and a C-terminal tail. The β-sheets of the N-terminal CK2α domains of the CK2 holoenzyme structure (Niefind et al. 2001) were drawn as black C_α traces after structural superimposition to indicate the location of the CK2α/CK2β interface. Two sulfate ions found at the juxta-dimer interface region are covered with FoFc-omit density (cutoff level 3 σ above the mean). The figure was drawn with BOBSCRIPT (Esnouf 1997) and Raster3D (Merrit and Bacon 1997). (B) Stereo picture of the C-terminal two-stranded β-sheet (β4β5) of a hsCK2β^1–193 monomer. For reasons of clarity some side chains were left out. The hsCK2β^1–193 structure segment is covered by electron density drawn with a contour level of 1 σ. The purple dotted lines indicate hydrogen bonds; distances are given in angstroms. For comparison the backbone of the equivalent region of the CK2 holoenzyme together with some side chains are drawn in black. The figure was prepared with BOBSCRIPT (Esnouf 1997) and Raster3D (Merrit and Bacon 1997). (C) Stereo picture of the anion binding sites at the juxta-dimer interface region. The sites are occupied by sulfate ions in the hsCK2β^1–193 structure, by phosphate ions (black) in the CK2 holoenzyme structure (Niefind et al. 2001), and by negatively charged side chains from a crystallographic neighbor (ball-and-stick representation of reduced size) in one of four dimers per asymmetric unit of the CK2β^2–182 structure (Bertrand et al. 2004). The sulfate ions are covered with blue, the protein parts of hsCK2β^1–193 with green pieces of electron density (all taken from the final 2Fo − Fc density map and contoured with a cutoff level of 1 σ). Some hydrogen bonds to the sulfate ions are indicated by dotted lines in magenta color. The figure was prepared with BOBSCRIPT (Esnouf 1997) and Raster3D (Merrit and Bacon 1997).