NMR Insights into the Conformational Dynamics and Binding Mechanism of the Cardiac Troponin Complex

Introduction: Cardiac Troponin (cTn) is a sarcomeric protein complex that plays a fundamental role in regulating the force generation of actomyosin mediated by Ca2+ in striated muscles. The three components of the complex are the Ca2+ binding sensor (cTnC), the inhibitory subunit, troponin I (cTnI), and the tropomyosin-binding subunit, troponin T (cTnT), which anchors the cTn complex to the thin filament. The structures of the regulatory domain of cardiac troponin (N-cTnC) bound to a Ca2+ show few structural changes compared to its apo form. However, the open conformation is observed in structures of N-cTnC bound to a Ca2+ and the switch peptide of cTnI (swcTnI). Objectives: The molecular recognition mechanism of swcTnI by N-cTnC remains an open question. To investigate the role of cTnC conformational dynamics, this work aims to characterize the thermodynamics and kinetics of the interaction between cTnC and swcTnI through nuclear magnetic resonance (NMR) spectroscopy. Methodology: 1H-15N HSQC and 1H-RD-CPMG experiments of 2H-15N-labeled cTnC with different swcTnI peptide concentrations were acquired. Structural changes in cTnC upon swcTnI titration were assessed through chemical shift perturbation (CSP) analysis. To gain further insights into the thermodynamics, kinetics, and molecular recognition mechanism of the interaction, NMR lineshape analysis was performed using the Matlab-based software Titan (Sci. Rep. v. 6, n. 1, p. 24826, 2016). Using inhouse Python scripts, CPMG experiments were also analyzed quantitatively to extract structural, kinetic, and thermodynamic information. Results: CSP analysis indicated that residues from the cTnC N-terminal domain were more affected by the interaction with swcTnI. Residues distant from the C-terminal domain were also perturbed, suggesting structural allostery triggered by the interaction. Lineshape analysis of the titration spectra indicated that a simple two-state binding mechanism adequately describes the cTnC-swcTnI interaction. Data fitting and spectra simulation were utilized to estimate the dissociation constant and off-rate of the cTnC-swcTnI interaction complex. This work was supported by CNPq, FAPEMIG, and CAPES. Further acknowledgments to the Laboratório de Ressonância Magnética de Alta Resolução da UFMG - LAREMAR