Dilated Cardiomyopathy Mutations and Phosphorylation disrupt the Active Orientation of Cardiac Troponin C.

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 N R relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R 10 s), being connected by a flexible linker. However, upon addition of cTnI, the complex tumbles as a rigid unit (R 30 s). 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 N R rates (R 17-23 s). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI and cTnI. Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI, cTnC-cTnT, or cTnC-cTnI-cTnT), 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.