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Spin‐labeled derivatives of cardiotonic steroids as tools for characterization of the extracellular entrance to the binding site on Na + ,K + ‐ ATP ase
Author(s) -
Guo JinHua,
Jiang RenWang,
Andersen Jacob Lauwring,
Esmann Mikael,
Fedosova Natalya U.
Publication year - 2018
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14480
Subject(s) - electron paramagnetic resonance , chemistry , binding site , steric effects , crystallography , spin label , ouabain , active site , site directed spin labeling , stereochemistry , docking (animal) , hyperfine structure , rotational correlation time , enzyme , nuclear magnetic resonance , sodium , biochemistry , medicine , physics , nursing , organic chemistry , quantum mechanics
The information obtained from crystallized complexes of the Na + ,K + ‐ ATP ase with cardiotonic steroids ( CTS ) is not sufficient to explain differences in the inhibitory properties of CTS such as stereoselectivity of CTS binding or effect of glycosylation on the preference to enzyme isoforms. The uncertainty is related to the spatial organization of the hydrophilic cavity at the entrance of the CTS ‐binding site. Therefore, there is a need to supplement the crystallographic description with data obtained in aqueous solution, where molecules have significant degree of flexibility. This work addresses the applicability of the electron paramagnetic resonance ( EPR ) method for the purpose. We have designed and synthesized spin‐labeled compounds based on the cinobufagin steroid core. The length of the spacer arms between the steroid core and the nitroxide group determines the position of the reporting group (N‐O) confined to the binding site. High affinity to Na + ,K + ‐ ATP ase is inferred from their ability to inhibit enzymatic activity. The differences between the EPR spectra in the absence and presence of high ouabain concentrations identify the signature peaks originating from the fraction of the spin labels bound within the ouabain site. The degree of perturbations of the EPR spectra depends on the length of the spacer arm. Docking of the compounds into the CTS site suggests which elements of the protein structure might be responsible for interference with the spin label (e.g., steric clashes or immobilization). Thus, the method is suitable for gathering information on the cavity leading to the CTS ‐binding site in Na + ,K + ‐ ATP ase in all conformations with high affinity to CTS .