RESUMEN
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 CK2ß, 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.
RESUMEN
Na+/K+-ATPase transports Na+ and K+ ions across the cell membrane via an ion-binding site becoming alternatively accessible to the intra- and extracellular milieu by conformational transitions that confer marked changes in ion-binding stoichiometry and selectivity. To probe the mechanism of these changes, we used molecular simulation and free-energy perturbation approaches to identify probable protonation states of Na+- and K+-coordinating residues in E1P and E2P conformations of Na+/K+-ATPase. Analysis of these simulations revealed a molecular mechanism responsible for the change in protonation state: the conformation-dependent binding of an anion (a chloride ion in our simulations) to a previously unrecognized cytoplasmic site in the loop between transmembrane helices 8 and 9, which influences the electrostatic potential of the crucial Na+-coordinating residue Asp926 This mechanistic model is consistent with experimental observations and provides a molecular-level picture of how E1P to E2P enzyme conformational transitions are coupled to changes in ion-binding stoichiometry and selectivity.