CK2β variants associated with Poirier–Bienvenu neurodevelopmental syndrome differentially affect CK2 holoenzyme assembly and stability

Introduction Mutations in the CSNK2B gene, which encodes the non-catalytic CK2β subunit of human protein kinase CK2, can cause Poirier -Bienvenu neurodevelopmental syndrome (POBINDS). The CK2 holoenzyme comprises two regulatory CK2β subunits and two catalytic CK2α or CK2α′ subunits. The interaction between CK2β and the catalytic CK2α/α’ subunits influences substrate phosphorylation and protein thermostability. Key structural regions involved in α/β interactions include the αF helix and hairpin loop, whereas β/β dimerization is stabilized by the zinc finger domain. In this study, we investigated 12 POBINDS-associated missense variants located within functional regions of CK2β to determine their effects on dimerization, stability, holoenzyme assembly, and kinase activity. Methods Kinase activity assays were used to assess the functional effects of the CK2β variants. Size-exclusion chromatography, activity assays and isothermal titration calorimetry were employed to investigate holoenzyme assembly and subunit interactions. Negative-stain electron microscopy was utilized to examine the structural integrity of selected holoenzymes. Differential scanning fluorimetry was performed to assess the thermostability of the variants. Results Most variants in the αF helix, hairpin loop, and zinc-finger domain significantly reduce holoenzyme kinase activity, even when CK2β is present in substantial excess. Size exclusion chromatography and isothermal titration calorimetry confirmed that these variants markedly impair holoenzyme formation. However, the zinc-finger variant C137G retains the ability to form intact holoenzymes, as further demonstrated by negative-stain electron microscopy reconstruction. Thermostability analyses using differential scanning fluorimetry further indicated that several disease-associated variants, particularly those with melting temperatures close to physiological temperature, may produce unstable CK2β subunits and thereby impair holoenzyme assembly in vivo. Discussion These findings demonstrate that POBINDS-associated CK2β variants can disrupt CK2 holoenzyme formation and stability, ultimately reducing CK2 activity through distinct molecular effects.