Double Quantum Coherence EPR Reveals the Structure–Function Relationships of the Cardiac Troponin C–Troponin I Complex Regulated by Ca2+ Ions and a Phosphomimetic

Jun Abe, Shoji Ueki, Seigo Yamauchi, Toshiaki Arata, Yasunori Ohba

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

Troponin (Tn) is a protein that consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT), and Tn controls cardiac muscle contraction by calcium ion binding and phosphorylation. The Ca2+-binding site is the E–F hand motif (C helix–loop–D helix) in the N-terminal domain of TnC, and the structural transition induced by Ca2+ is the opening of these helices and the interaction with TnI, probably at the A and B helices. In this paper, we studied structural changes in the TnC–TnI binary complex on Ca2+ binding by double quantum coherence (DQC) distance measurements. We used a binary complex of the cardiac troponin C and I (cTnC and cTnI) complexes, chose four positions of nitroxide spin label at helices A, B, C, and D in the N-terminal domain and chose the E helix in the C-terminal domain as the reference position to study the structural changes on Ca2+ addition. The label positions were (A22C/S98C), (M47C/S98C), (Q58C/S98C), and (C84/S98C) for the A, B, C, and D helices, respectively. The effects of phosphorylation of the cardiac-specific N-terminal region of cTnI were studied using a phosphomimetic cTnI mutant. Analysis of the modulation of the DQC echo signals provided the distribution of the spin–spin distance. The distances averaged over the distribution showed that the labels on the A, B, and C helices decreased, i.e., moved to the E helix, on Ca2+ binding, while the distance of the label on the D helix showed almost no change. Shoulders and/or small separate peaks were observed in the shape of the distribution and were analyzed as the sum of a few Gaussian functions. The Gaussian functions were grouped into two components, components 1 and 2, at the longer and shorter distances, respectively, separated by 0.7–1.5 nm. The fractions of component 2 were ca. 0.1–0.2 in the Ca2+-free state and increased by 0.2–0.3 on Ca2+ addition, suggesting that the increase in component 2 is related to physiological control of cardiac muscle contraction. The phosphomimetic-modification effects on the Ca2+-induced changes of the fraction of components and the distances of the C- and D-helix labels are small. On the other hand, in the A and B helices, there are significant effects on the Ca2+-induced changes in the distances of the components. The different behaviors of A/B and C/D helices support the current model of the phosphorylation effects in which both N-terminal region and regulatory domain of cTnI interact with the A and B helices of cTnC.

Original languageEnglish
Pages (from-to)893-910
Number of pages18
JournalApplied Magnetic Resonance
Volume49
Issue number8
DOIs
Publication statusPublished - 2018 Aug 1

ASJC Scopus subject areas

  • Atomic and Molecular Physics, and Optics

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