Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca2+ dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca2+ binding in the NH2-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin's detailed dynamics, troponin's regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca2+ slowed HDX up to 2 orders of magnitude within the NH2-lobe and the NH2-lobe-associated TnI switch helix, implying that Ca2+ greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca2+ and actin. Ca2+-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca2+ destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca2+-mediated regulation: the coiled-coil's NH2-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca2+-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca2+, including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca2+ release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin's dynamic features.