Spin-labeled derivatives of cardiotonic steroids as tools for characterization of the extracellular entrance to the binding site on Na+,K+-ATPase

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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+-ATPase. / Guo, Jin-Hua ; Jiang, Ren-Wang; Andersen, Jacob Lauwring; Esmann, Mikael; Fedosova, Natalya.

I: F E B S Journal, Bind 285, Nr. 12, 2018, s. 2292-2305.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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@article{193b339385f248f6a624f5d29bc06572,
title = "Spin-labeled derivatives of cardiotonic steroids as tools for characterization of the extracellular entrance to the binding site on Na+,K+-ATPase",
abstract = "The information obtained from crystallized complexes of the Na + ,K + -ATPase 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 + -ATPase 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 + -ATPase in all conformations with high affinity to CTS. ",
keywords = "EPR, Na pump, Structure, membrane protein, protein–ligand interactions",
author = "Jin-Hua Guo and Ren-Wang Jiang and Andersen, {Jacob Lauwring} and Mikael Esmann and Natalya Fedosova",
year = "2018",
doi = "10.1111/febs.14480",
language = "English",
volume = "285",
pages = "2292--2305",
journal = "F E B S Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell Publishing Ltd.",
number = "12",

}

RIS

TY - JOUR

T1 - Spin-labeled derivatives of cardiotonic steroids as tools for characterization of the extracellular entrance to the binding site on Na+,K+-ATPase

AU - Guo, Jin-Hua

AU - Jiang, Ren-Wang

AU - Andersen, Jacob Lauwring

AU - Esmann, Mikael

AU - Fedosova, Natalya

PY - 2018

Y1 - 2018

N2 - The information obtained from crystallized complexes of the Na + ,K + -ATPase 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 + -ATPase 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 + -ATPase in all conformations with high affinity to CTS.

AB - The information obtained from crystallized complexes of the Na + ,K + -ATPase 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 + -ATPase 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 + -ATPase in all conformations with high affinity to CTS.

KW - EPR

KW - Na pump

KW - Structure

KW - membrane protein

KW - protein–ligand interactions

U2 - 10.1111/febs.14480

DO - 10.1111/febs.14480

M3 - Journal article

VL - 285

SP - 2292

EP - 2305

JO - F E B S Journal

JF - F E B S Journal

SN - 1742-464X

IS - 12

ER -