Journal: Frontiers in Molecular Biosciences

Article Title: Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2α Lys198Arg

doi: 10.3389/fmolb.2022.831693

Figure Lengend Snippet: ITC characterization of the effect of the Lys198Arg mutation on the CK2α/CK2β interaction. (A/B) Representative direct and integrated ITC curves for the interaction of CK2β 1-193 with CK2α 1-335 (A) and CK2α 1-335,Lys198Arg (B) generated with ORIGIN. (C) Thermodynamic data after processing of ITC curves. The numbers represent mean values plus corresponding standard deviations for triplicate measurements.

Article Snippet: Four different CK2 substrate peptides (RRRDDDSDDD, RRRDDDTDDD, RRRDDDSGGD, and RRREDEYDDD) were purchased from GenScript (Leiden, Netherlands).

Techniques: Mutagenesis, Generated

Journal: Frontiers in Molecular Biosciences

Article Title: Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2α Lys198Arg

doi: 10.3389/fmolb.2022.831693

Figure Lengend Snippet: Characterization of the effect of the Lys198Arg mutation on the CK2α/CK2β interaction using a fluorescence-based assay. The fluorescence of CK2β 1-193 -DBCO-Sulfo-Cy5 quenched by increasing concentrations of CK2α (A) or CK2α Lys198Arg (B) in the range from 0.3 to 5,000 nM was measured. The binding curves were generated and processed using the software MO. Affinity Analysis v2.1.3 (Nanotemper, München, Germany). The K D values given in the graphs are averages from three independent experiments.

Article Snippet: Four different CK2 substrate peptides (RRRDDDSDDD, RRRDDDTDDD, RRRDDDSGGD, and RRREDEYDDD) were purchased from GenScript (Leiden, Netherlands).

Techniques: Mutagenesis, Fluorescence, Binding Assay, Generated, Software

Journal: Frontiers in Molecular Biosciences

Article Title: Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2α Lys198Arg

doi: 10.3389/fmolb.2022.831693

Figure Lengend Snippet: Differential scanning fluorimetry to determine the thermal stability of the CK2α variants of this study and its change by CK2β 1-193 . (A/B) Negative first derivatives of representative melting curves of either full-lengths CK2α variants (A) or C-terminally truncated CK2α variants (B) in the absence of CK2β 1-193 (solid blue and red lines) and in the presence of CK2β 1–193 (dashed blue and red lines). For comparison, the curve measured with unbound CK2β 1–193 is drawn as black solid line in both panels. (C) Melting temperatures derived from DSF curves as visible in panels A and B. Averages and standard deviations of three independent measurements, respectively, are given.

Article Snippet: Four different CK2 substrate peptides (RRRDDDSDDD, RRRDDDTDDD, RRRDDDSGGD, and RRREDEYDDD) were purchased from GenScript (Leiden, Netherlands).

Techniques: Comparison, Derivative Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2α Lys198Arg

doi: 10.3389/fmolb.2022.831693

Figure Lengend Snippet: X-ray data and refinement statistics of a CK2α Lys198Arg structure.

Article Snippet: Four different CK2 substrate peptides (RRRDDDSDDD, RRRDDDTDDD, RRRDDDSGGD, and RRREDEYDDD) were purchased from GenScript (Leiden, Netherlands).

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2α Lys198Arg

doi: 10.3389/fmolb.2022.831693

Figure Lengend Snippet: Crystal structure of CK2α Lys198Arg . All depicted pieces of electron density were extracted from the final 2F o -F c map using a contouring level of 1 σ. The figure was prepared with . (A/B) Global overview of one CK2α Lys198Arg protomer, illustrating either the electrostatic surface with bound sulfate ions (A) or the secondary structure elements (B) . (C) Details of the anion binding sites at the activation loop (with bound P+3 sulfate) and at the P+1 loop. A substrate peptide modelled into the active site as described by is shown with light-blue carbon atoms. Sulfate ions or a heparin sulfo group from several other wild-type-like CK2α structures ( ; ; ) as well as the essential part of the P+1 loop in the MAP kinase p38γ were drawn for comparison. (D/E) The P+1 loop in chain A (D) or in chain B (E) of the CK2α Lys198Arg structure; in both cases, the P+1 loop in the wild-type-like CK2α 1-335 structure 2PVR plus the bound sulfate ion were drawn to illustrate changes in the backbone and the precise sulfate ion location.

Article Snippet: Four different CK2 substrate peptides (RRRDDDSDDD, RRRDDDTDDD, RRRDDDSGGD, and RRREDEYDDD) were purchased from GenScript (Leiden, Netherlands).

Techniques: Binding Assay, Activation Assay, Comparison

Journal: Frontiers in Molecular Biosciences

Article Title: Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2α Lys198Arg

doi: 10.3389/fmolb.2022.831693

Figure Lengend Snippet: Michaelis-Menten kinetics with varying substrate concentrations to examine changes in substrate specificity caused by the CK2α mutation Lys198Arg. | (A/C/E) Lineweaver-Burk plots for wild-type (CK2α) 2 (CK2β) 2 and the substrates RRRDDDSDDD (A) , RRRDDDSGGD (C) , or RRRDDDTDDD (E) . (B/D/F) Lineweaver-Burk plots for the mutant (CK2α Lys198Arg ) 2 (CK2β) 2 and the substrates RRRDDDSDDD (B) , RRRDDDSGGD (D) , or RRRDDDTDDD (F) . (G) Overview of the enzymological data derived from the Lineweaver-Burk plots in panels (A–F) . The catalytic efficiency values (k cat /K M ) are highlighted in red colour.

Article Snippet: Four different CK2 substrate peptides (RRRDDDSDDD, RRRDDDTDDD, RRRDDDSGGD, and RRREDEYDDD) were purchased from GenScript (Leiden, Netherlands).

Techniques: Mutagenesis, Derivative Assay