Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2αLys198Arg

Abstract

Specific de novo mutations in the CSNK2A1 gene, which encodes CK2α, the catalytic subunit of protein kinase CK2, are considered as causative for the Okur-Chung neurodevelopmental syndrome (OCNDS). OCNDS is a rare congenital disease with a high phenotypic diversity ranging from neurodevelopmental disabilities to multi-systemic problems and characteristic facial features. A frequent OCNDS mutation is the exchange of Lys198 to Arg at the center of CK2α′s P+1 loop, a key element of substrate recognition. According to preliminary data recently made available, this mutation causes a significant shift of the substrate specificity of the enzyme. We expressed the CK2αLys198Arg recombinantly and characterized it biophysically and structurally. Using isothermal titration calorimetry (ITC), fluorescence quenching and differential scanning fluorimetry (Thermofluor), we found that the mutation does not affect the interaction with CK2β, the non-catalytic CK2 subunit, and that the thermal stability of the protein is even slightly increased. However, a CK2αLys198Arg crystal structure and its comparison with wild-type structures revealed a significant shift of the anion binding site harboured by the P+1 loop. This observation supports the notion that the Lys198Arg mutation causes an alteration of substrate specificity which we underpinned here with enzymological data.

Predictive functional, statistical and structural analysis of CSNK2A1 and CSNK2B variants linked to neurodevelopmental diseases

“…Residues not in the interface were not predicted to change the affinity for CK2β. This hypothesis is supported by the literature, at least in the case of Lys198Arg (Werner et al, 2022).…”

“…For the mutations identified, potential mechanisms of action include: 1) change of substrate specificity but still a strong overlap with canonical CK2 substrates leading to phosphorylation of non-canonical substrates, as is likely the case for Lys198Arg as supported by the literature (Caefer et al, 2022;Werner et al, 2022); 2) change in substrate specificity with no overlap to CK2 canonical substrates (e.g., Lys198 exchange to Thr, Gln, Glu Asn, Ile) which we hypothesize is deleterious as proteins will be phosphorylated in an unregulated way; 3) interference with substrate phosphorylation; 4) the global fold of CK2α is disrupted whenever the amino acid is involved in stabilizing part of the architecture (e.g., Arg312 mutations). We do not expect a loss on the ability to bind substrates, as substrate recognition is not determined by one single residue.…”

“…Lys198 was described as the key residue determining the anion binding potential of the P+1 loop ( Sarno et al, 1995 ; Sarno et al 1996 ; Sarno et al 1997 ). The crystal structure of the CK2α Lys198Arg variant discloses a significant shift of a sulfate ion marking the anion binding site in the P+1 position ( Werner et al, 2022 ). This observation supports the notion by Caefer et al that the Lys198Arg mutation causes an alteration of substrate specificity ( Caefer et al, 2022 ).…”

“…There is also experimental evidence that one CK2β mutant has low expression levels and another does not interact with CK2α ( Nakashima et al, 2019 ). Recently, altered substrate specificity has been reported for CK2α variant K198R ( Caefer et al, 2022 ; Werner et al, 2022 ). However, we still have no knowledge of the signaling, cellular and biological mechanism of action of the mutations in CK2α and CK2β that are associated with OCNDS and POBINDS, respectively.…”

A Case of Okur-Chung Neurodevelopmental Syndrome with a Novel, de novo Variant on the <i>CSNK2A1</i> Gene in a Turkish Patient

“…OCNDS is a rare congenital autosomal dominant disorder that is observed with different phenotypic signs and a characteristic facial structure [Werner et al, 2022]. Moreover, it ranges in its presentation from neurodevelopmental disabilities to multisystem disorders.…”