Abstract
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.
Highlights
Ca2ϩ binding to the thin filament protein troponin strictly regulates muscle contraction
Troponin causes cessation of cardiac contraction when Ca2ϩ is dissociated from one specific site: site II in the troponin C (TnC) subunit’s NH2-lobe
The location of the graphed peptide within troponin is shown in black in a cropped thumbnail image. (Fig. 5A shows the same orientation with annotations.) The faster TnC transitions for CBMII troponin in each panel, compared with hydrogen-deuterium exchange (HDX) for unmodified troponin, indicate the absence of a large stabilizing effect that occurs when site II is occupied by Ca2ϩ
Summary
Ca2ϩ binding to the thin filament protein troponin strictly regulates muscle contraction. 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. Contraction is strictly dependent upon Ca2ϩ binding to TnC To investigate this central, partially understood (8 –14) regulatory mechanism, the present study widely examines and quantitatively maps the effects of regulatory site II Ca2ϩ on cardiac troponin’s dynamics. Effects of Regulatory Site Ca2؉ on Troponin Dynamics to slow the intrinsic HDX rate, it was possible to characterize many troponin residues for the first time and to assess others very much better than previously. These lower temperature conditions are used to map the effect of regulatory site Ca2ϩ on troponin dynamics. The data comprehensively and quantitatively validate the concept that cardiomyopathy-inducing mutations cluster in dynamic regions of troponin
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
