Significance of troponin dynamics for Ca2+-mediated regulation of contraction and inherited cardiomyopathy.
Ca(2+) dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca(2+) binding in the NH(2)-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. Ca(2+) slowed HDX up to 2 orders of magnitude within the NH(2)-lobe and the NH(2)-lobe-associated TnI switch helix, implying that Ca(2+) 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 Ca(2+) and actin. Ca(2+)-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. Ca(2+) destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca(2+)-mediated regulation: the coiled-coil's NH(2)-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 Ca(2+)-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca(2+), including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca(2+) 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.
Department of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
Devanand
D
Kowlessur
Larry S
LS
Tobacman
D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D009202:Cardiomyopathies; D041961:Deuterium Exchange Measurement; D030342:Genetic Diseases, Inborn; D006801:Humans; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D019209:Troponin C; D019210:Troponin I; D002118:Calcium