Binding of levosimendan, a calcium sensitizer, to cardiac troponin C

Tia Sorsa, Sami Heikkinen, M. Bret Abbott, Ekram Abusamhadneh, Tero Laakso, Carola Tilgmann, Ritva Serimaa, Arto Annila, Paul Rosevear, Torbjörn Drakenberg, Piero Pollesello, Ilkka Kilpeläinen

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Abstract

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.
Original languageEnglish
Pages (from-to)9337-9343
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number12
DOIs
Publication statusPublished - 2001
MoE publication typeA1 Journal article-refereed

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Troponin C
Calcium
Nuclear magnetic resonance
Sodium Azide
X-Rays
Scattering
Biomolecular Nuclear Magnetic Resonance
simendan
Contractile Proteins
X rays
Dithiothreitol
Reducing Agents
Serine
Cysteine
Proteins
Experiments
Binding Sites

Cite this

Sorsa, T., Heikkinen, S., Abbott, M. B., Abusamhadneh, E., Laakso, T., Tilgmann, C., ... Kilpeläinen, I. (2001). Binding of levosimendan, a calcium sensitizer, to cardiac troponin C. Journal of Biological Chemistry, 276(12), 9337-9343. https://doi.org/10.1074/jbc.M007484200
Sorsa, Tia ; Heikkinen, Sami ; Abbott, M. Bret ; Abusamhadneh, Ekram ; Laakso, Tero ; Tilgmann, Carola ; Serimaa, Ritva ; Annila, Arto ; Rosevear, Paul ; Drakenberg, Torbjörn ; Pollesello, Piero ; Kilpeläinen, Ilkka. / Binding of levosimendan, a calcium sensitizer, to cardiac troponin C. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 12. pp. 9337-9343.
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title = "Binding of levosimendan, a calcium sensitizer, to cardiac troponin C",
abstract = "Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.",
author = "Tia Sorsa and Sami Heikkinen and Abbott, {M. Bret} and Ekram Abusamhadneh and Tero Laakso and Carola Tilgmann and Ritva Serimaa and Arto Annila and Paul Rosevear and Torbj{\"o}rn Drakenberg and Piero Pollesello and Ilkka Kilpel{\"a}inen",
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Sorsa, T, Heikkinen, S, Abbott, MB, Abusamhadneh, E, Laakso, T, Tilgmann, C, Serimaa, R, Annila, A, Rosevear, P, Drakenberg, T, Pollesello, P & Kilpeläinen, I 2001, 'Binding of levosimendan, a calcium sensitizer, to cardiac troponin C', Journal of Biological Chemistry, vol. 276, no. 12, pp. 9337-9343. https://doi.org/10.1074/jbc.M007484200

Binding of levosimendan, a calcium sensitizer, to cardiac troponin C. / Sorsa, Tia; Heikkinen, Sami; Abbott, M. Bret; Abusamhadneh, Ekram; Laakso, Tero; Tilgmann, Carola; Serimaa, Ritva; Annila, Arto; Rosevear, Paul; Drakenberg, Torbjörn; Pollesello, Piero; Kilpeläinen, Ilkka.

In: Journal of Biological Chemistry, Vol. 276, No. 12, 2001, p. 9337-9343.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Binding of levosimendan, a calcium sensitizer, to cardiac troponin C

AU - Sorsa, Tia

AU - Heikkinen, Sami

AU - Abbott, M. Bret

AU - Abusamhadneh, Ekram

AU - Laakso, Tero

AU - Tilgmann, Carola

AU - Serimaa, Ritva

AU - Annila, Arto

AU - Rosevear, Paul

AU - Drakenberg, Torbjörn

AU - Pollesello, Piero

AU - Kilpeläinen, Ilkka

PY - 2001

Y1 - 2001

N2 - Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

AB - Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

U2 - 10.1074/jbc.M007484200

DO - 10.1074/jbc.M007484200

M3 - Article

VL - 276

SP - 9337

EP - 9343

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 12

ER -

Sorsa T, Heikkinen S, Abbott MB, Abusamhadneh E, Laakso T, Tilgmann C et al. Binding of levosimendan, a calcium sensitizer, to cardiac troponin C. Journal of Biological Chemistry. 2001;276(12):9337-9343. https://doi.org/10.1074/jbc.M007484200