De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

CSNK2B encodes for casein kinase II subunit beta (CK2b), the regulatory subunit of casein kinase II (CK2), which is known to mediate diverse cellular pathways. Variants in this gene have been recently identified as a cause of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), but functional evidence is sparse. Here, we report five unrelated individuals: two of them manifesting POBINDS, while three are identified to segregate a new intellectual disability-craniodigital syndrome (IDCS), distinct from POBINDS. The three IDCS individuals carried two different de novo missense variants affecting the same codon of CSNK2B. Both variants, NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn, lead to an upregulation of CSNK2B expression at transcript and protein level, along with global dysregulation of canonical Wnt signaling. We found impaired interaction of the two key players DVL3 and b-catenin with mutated CK2b. The variants compromise the kinase activity of CK2 as evident by a marked reduction of phosphorylated b-catenin and consequent absence of active b-catenin inside nuclei of the patient-derived lymphoblastoid cell lines (LCLs). In line with these findings, whole-transcriptome profiling of patient-derived LCLs harboring the NP_001311.3; p.Asp32His variant confirmed a marked difference in expression of genes involved in the Wnt signaling pathway. In addition, whole-phosphoproteome analysis of the LCLs of the same subject showed absence of phosphorylation for 313 putative CK2 substrates, enriched in the regulation of nuclear b-catenin and transcription of the target genes. Our findings suggest that discrete variants in CSNK2B cause dominant-negative perturbation of the canonical Wnt signaling pathway, leading to a new craniodigital syndrome distinguishable from POBINDS

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

Okur-Chung Neurodevelopmental Syndrome (OCNDS) and Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS) were recently identified as rare neurodevelopmental disorders. OCNDS and POBINDS are associated with heterozygous mutations in the CSNK2A1 and CSNK2B genes which encode CK2 alpha, a serine/threonine protein kinase, and CK2 beta, a regulatory protein, respectively, which together can form a tetrameric enzyme called protein kinase CK2. A challenge in OCNDS and POBINDS is to understand the genetic basis of these diseases and the effect of the various CK2? and CK2 beta mutations. In this study we have collected all variants available to date in CSNK2A1 and CSNK2B, and identified hotspots. We have investigated CK2? and CK2 beta missense mutations through prediction programs which consider the evolutionary conservation, functionality and structure or these two proteins, compared these results with published experimental data on CK2 alpha and CK2 beta mutants, and suggested prediction programs that could help predict changes in functionality of CK2 alpha mutants. We also investigated the potential effect of CK2 alpha and CK2 beta mutations on the 3D structure of the proteins and in their binding to each other. These results indicate that there are functional and structural consequences of mutation of CK2 alpha and CK2 beta, and provide a rationale for further study of OCNDS and POBINDS-associated mutations. These data contribute to understanding the genetic and functional basis of these diseases, which is needed to identify their underlying mechanisms

Structural and Enzymological Evidence for an Altered Substrate Specificity in Okur-Chung Neurodevelopmental Syndrome Mutant CK2 alpha(Lys198Arg)

Specific de novo mutations in the CSNK2A1 gene, which encodes CK2 alpha, 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 CK2 alpha ' 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 CK2 alpha(Lys198Arg) 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 CK2 beta, the non-catalytic CK2 subunit, and that the thermal stability of the protein is even slightly increased. However, a CK2 alpha(Lys198Arg) 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

Crystal structure of highly glycosylated human leukocyte elastase in complex with an S2 ' site binding inhibitor

Glycosylated human leukocyte elastase (HLE) was crystallized and structurally analysed in complex with a 1,3-thiazolidine-2,4-dione derivative that had been identified as an HLE inhibitor in preliminary studies. In contrast to previously described HLE structures with small-molecule inhibitors, in this structure the inhibitor does not bind to the Si and S2 substrate-recognition sites; rather, this is the first HLE structure with a synthetic inhibitor in which the S2' site is blocked that normally binds the second side chain at the C-terminal side of the scissile peptide bond in a substrate protein. The inhibitor also induces the formation of crystalline HLE dimers that block access to the active sites and that are also predicted to be stable in solution. Neither such HLE dimers nor the corresponding crystal packing have been observed in previous HLE crystal structures. This novel crystalline environment contributes to the observation that comparatively large parts of the N-glycan chains of HLE are defined by electron density. The final HLE structure contains the largest structurally defined carbohydrate trees among currently available HLE structures

Unexpected Binding Mode of a Potent Indeno[1,2-b]indole-Type Inhibitor of Protein Kinase CK2 Revealed by Complex Structures with the Catalytic Subunit CK2α and Its Paralog CK2α′

Protein kinase CK2, a member of the eukaryotic protein kinase superfamily, is associated with cancer and other human pathologies and thus an attractive drug target. The indeno[1,2-b]indole scaffold is a novel lead structure to develop ATP-competitive CK2 inhibitors. Some indeno[1,2-b]indole-based CK2 inhibitors additionally obstruct ABCG2, an ABC half transporter overexpressed in breast cancer and co-responsible for drug efflux and resistance. Comprehensive derivatization studies revealed substitutions of the indeno[1,2-b]indole framework that boost either the CK2 or the ABCG2 selectivity or even support the dual inhibition potential. The best indeno[1,2-b]indole-based CK2 inhibitor described yet (IC50 = 25 nM) is 5-isopropyl-4-(3-methylbut-2-enyl-oxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p). Herein, we demonstrate the membrane permeability of 4p and describe co-crystal structures of 4p with CK2α and CK2α′, the paralogs of human CK2 catalytic subunit. As expected, 4p occupies the narrow, hydrophobic ATP site of CK2α/CK2α′, but surprisingly with a unique orientation: its hydrophobic substituents point towards the solvent while its two oxo groups are hydrogen-bonded to a hidden water molecule. An equivalent water molecule was found in many CK2α structures, but never as a critical mediator of ligand binding. This unexpected binding mode is independent of the interdomain hinge/helix αD region conformation and of the salt content in the crystallization medium

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2alpha/CK2beta interaction

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2 alpha and CK2 beta in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2 alpha(1-335) and the fluorescent probe CF-Ahx-Pc as a CK2 beta analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2 alpha(1-335). Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2 alpha-binding motif of CK2 beta. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2 alpha(1-335) complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2 alpha(1-335) were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a K-D value of 240 nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2 beta, with an IC50 value of 92 mu M. (C) 2014 Elsevier Inc. All rights reserved

Diacritic Binding of an Indenoindole Inhibitor by CK2 alpha Paralogs Explored by a Reliable Path to Atomic Resolution CK2 alpha ' Structures

CK2 alpha and CK2 alpha' are the two isoforms of the catalytic subunit of human protein kinase CK2, an important target for cancer therapy. They have similar, albeit not identical functional and structural properties, and were occasionally reported to be inhibited with distinct efficacies by certain ATP-competitive ligands. Here, we present THN27, an indeno[1,2-b] indole derivative, as a further inhibitor with basal isoform selectivity. The selectivity disappears when measured using CK2 alpha/CK2 alpha' complexes with CK2 beta, the regulatory CK2 subunit. Co-crystal structures of THN27 with CK2 alpha and CK2 alpha' reveal that subtle differences in the conformational variability of the inter-domain hinge region are correlated with the observed effect. In the case of CK2 alpha', a crystallographically problematic protein so far, this comparative structural analysis required the development of an experimental strategy that finally enables atomic resolution structure determinations with ab initio phasing of potentially any ATP-competitive CK2 inhibitor and possibly many non-ATP-competitive ligands as well bound to CK2 alpha'

Isoform specific inhibition of human protein kinase CK2α and CK2α’ by an indenoindole derivative

International audienc