Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Shibazaki, C.; Arai, Shigeki; Shimizu, R; Saeki, Morihisa; Kinoshita, Takayoshi; Ostermann, Andreas; Schrader, Tobias E.; Kurosaki, Yasuo; Sunami, Tomoko; Kuroki, Ryota; Adachi, Motoyasu

doi:10.1016/j.jmb.2018.09.018

Cited by 6 publications

(7 citation statements)

References 49 publications

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“…In the context of this study, two sulfate ions located at the “central substrate-recognition region” ( Figure 4A ) are particularly relevant because their binding sites are the P+1 loop, which harbors the critical Lys198Arg mutation, and the activation loop ( Figure 4B ). At these two cavities, sulfate ions have been found several times before ( Figure 4C ) ( Niefind et al, 2007 ; Shibazaki et al, 2018 ; Schnitzler and Niefind, 2021 ); in a recent high-resolution structure of a CK2α 1–335 /heparin complex, one of them—the P+1 loop site—even harbors a sulfo moiety of a heparin disulfo-glucosamine residue ( Figure 4C ) ( Schnitzler and Niefind, 2021 ). The most striking feature of the CK2α Lys198Arg /sulfate complex structure is that the position of the sulfate ion at the activation loop, which is designated as “P+3 sulfate” in Figure 4A/B/C for reasons explained below, is identical compared to the wild-type structures while the sulfate ion at the mutated P+1 loop was shifted by 3.5 Å in the direction of Arg195, one of the selectivity determinants of the P+1 loop ( Figure 4C ).…”

Section: Resultsmentioning

confidence: 93%

“…A substrate peptide modelled into the active site as described by Niefind et al (2007) is shown with light-blue carbon atoms. Sulfate ions or a heparin sulfo group from several other wild-type-like CK2α structures ( Niefind et al, 2007 ; Shibazaki et al, 2018 ; Schnitzler and Niefind, 2021 ) as well as the essential part of the P+1 loop in the MAP kinase p38γ ( Bellon et al, 1999 ) 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 ( Niefind et al, 2007 ) plus the bound sulfate ion were drawn to illustrate changes in the backbone and the precise sulfate ion location.…”

Section: Resultsmentioning

confidence: 99%

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

Werner

Gast

Lindenblatt

et al. 2022

Front. Mol. Biosci.

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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.

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Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

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

Werner

Gast

Lindenblatt

et al. 2022

Front. Mol. Biosci.

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“…To further explore the potential of CK2 regulation by copper, we took advantage of an in-silico approach: the Metal Ion-Binding Site Prediction and Docking Server ( Lin et al, 2016 ). Through analysis of various CK2α crystal structures in the Protein Data Bank (PDB) ( Kinoshita et al, 2011 ; Kinoshita et al, 2013 ; Shibazaki et al, 2018 ), multiple residues predicted to be implicated in copper binding were identified ( Figure 1B ). The majority of these residues are located within the catalytic domain of CK2α and are identical to the residues identified by homology analysis with MEK1.…”

Section: Resultsmentioning

confidence: 99%

“…The blue letters represent residues in CK2α that share homology with MEK1 at copper-binding residues. (B) Prediction output by the MIB site prediction server ( Lin et al, 2016 ) using three PDB files: 3war, 3at2, and 5zn0 ( Kinoshita et al, 2011 ; Kinoshita et al, 2013 ; Shibazaki et al, 2018 ). Residues were only included if they were predicted to bind copper across all three structures.…”

Section: Resultsmentioning

confidence: 99%

Copper Modulates the Catalytic Activity of Protein Kinase CK2

Chojnowski

Tsang

et al. 2022

Front. Mol. Biosci.

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Casein kinase 2 (CK2) is an evolutionarily conserved serine/threonine kinase implicated in a wide range of cellular functions and known to be dysregulated in various diseases such as cancer. Compared to most other kinases, CK2 exhibits several unusual properties, including dual co-substrate specificity and a high degree of promiscuity with hundreds of substrates described to date. Most paradoxical, however, is its apparent constitutive activity: no definitive mode of catalytic regulation has thus far been identified. Here we demonstrate that copper enhances the enzymatic activity of CK2 both in vitro and in vivo. We show that copper binds directly to CK2, and we identify specific residues in the catalytic subunit of the enzyme that are critical for copper-binding. We further demonstrate that increased levels of intracellular copper result in enhanced CK2 kinase activity, while decreased copper import results in reduced CK2 activity. Taken together, these findings establish CK2 as a copper-regulated kinase and indicate that copper is a key modulator of CK2-dependent signaling pathways.

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“…On the other hand, high conservation of protein structure around site B in the active state, especially the preservation of two flanking, bulky aromatic residues, may suggest the importance of buried solvent region for enzymatic function. Possible roles may include shaping conformational equilibrium of protein substrate binding region, 42 or participating in the extraction of a proton released out of the catalytic site during substrate phosphorylation 73. The latter possibility may involve well localized water molecules W1 and W2 (Figure 5A) that together participate in a hydrogen bond network that connects the catalytic aspartic acid (D166) with well conserved D220 from the F-helix.W2 water molecule is universally present within the site B in active and inactive catalytic subunits, and may also play a distinct structural role.…”

mentioning

confidence: 99%

Conserved internal hydration motifs in protein kinases

Setny

2020

Proteins

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Crystal structures of diverse protein kinase catalytic subunits reveal a number of water molecules whose positions within the protein core are better preserved than amino acid types in many functionally important locations. It remains unknown whether they play any particular role, and whether their removal, disturbing local interaction patterns to no smaller degree than amino acid mutations, can affect kinase stability and function. In this study, we apply an array of computational approaches to characterize hydration of kinase catalytic subunits. First, we systematically screen multiple crystal structures with the use of a simplified hydration model in order to determine the distribution of internal solvent and the degree of its conservation. Second, we analyze water structure, dynamics and binding affinity to buried hydration sites in a number of kinases, also taking into account their variable functional state. We find that a large portion of buried solvent is dynamic, possibly contributing to kinase conformational changes related to the activation process. In turn, binding free energies of some of tightly bound conserved water molecules to different kinases tend to shift in a similar manner following the change of their functional state. This finding highlights the likely specific role of internal solvent in fine tuning local protein plasticity.

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Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Cited by 6 publications

References 49 publications

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

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

Copper Modulates the Catalytic Activity of Protein Kinase CK2

Conserved internal hydration motifs in protein kinases

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