Cardiac troponin (cTn) is made up of three subunits, cTnC, cTnI, and cTnT. The regulatory N-terminal domain of cTnC (cNTnC) controls cardiac muscle contraction in a calcium-dependent manner. We show that calcium-saturated cNTnC can adopt two different orientations, with the "active" orientation consistent with the 2020 cryo-EM structure of the activated cardiac thin filament by Yamada et al. Using solution NMR ¹⁵N R₂ relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R₂ 10 s^-1), being connected by a flexible linker. However, upon addition of cTnI_1-77, the complex tumbles as a rigid unit (R₂ 30 s^-1). cTnI phosphomimetic mutants S22D/S23D, S41D/S43D and dilated cardiomyopathy- (DCM-)associated mutations cTnI K35Q, cTnC D75Y, and cTnC G159D destabilize the active orientation of cNTnC, with intermediate ¹⁵N R₂ rates (R₂ 17-23 s^-1). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI_1-37 and cTnI_135-209. Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI_38-134, cTnC-cTnT_223-288, or cTnC-cTnI_38-134-cTnT_223-288), the cNTnC domain is still immobilized, revealing a new interaction between cNTnC and the IT-arm that stabilizes a "dormant" orientation. We propose that the calcium sensitivity of the cardiac troponin complex is regulated by an equilibrium between active and dormant orientations, which can be shifted through post-translational modifications or DCM-associated mutations.

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