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-15 R-2 relaxation analysis, we demonstrate that the two domains of cTnC tumble independently (average R-2 10 s(-1)), being connected by a flexible linker. However, upon addition of cTnI(1-77), the complex tumbles as a rigid unit (R-2 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 N-15 R-2 rates (R-2 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. (C) 2021 The Author(s). Published by Elsevier Ltd.

Automated summary

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.