Dilated Cardiomyopathy Mutations and Phosphorylation disrupt the Active Orientation of Cardiac Troponin C. Issue 13 (25th June 2021)

• Record Type: Journal Article
• Authors: Mahmud, Zabed; Dhami, Prabhpaul S.; Rans, Caleb; Liu, Philip B.; Hwang, Peter M.
• Is Part Of: Journal of molecular biology. Volume 433:Issue 13(2021)
• Publication Date: 2021-06-25
• Subjects: nuclear magnetic resonance spectroscopy -- protein domain orientation -- intrinsically disordered region -- heritable cardiomyopathy -- systolic heart failure; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2021.167010
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: The cardiac troponin complex can adopt an active or dormant orientation. Competing orientations of troponin regulate cardiac muscle calcium sensitivity. Phosphomimetic cTnI mutations destabilize the active orientation. Some DCM-associated troponin mutations destabilize the active orientation. Abstract: 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 15 N 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 cTnI1-77, 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 15 N R2 rates (R2 17–23 s −1 ). The active orientation of cNTnC is stabilized by the flexible tails of cTnI, cTnI1-37 and cTnI135-209 . Surprisingly, when cTnC is incorporated into complexes lacking these tails (cTnC-cTnI38-134, cTnC-cTnT223-288, or cTnC-cTnI38-134 -cTnT223-288 ), 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.