title: Effects of HCM cTnI mutation R145G on troponin structure and modulation by PKA phosphorylation elucidated by molecular dynamics simulations.
abstract: Cardiac troponin (cTn) is a key molecule in the regulation of human cardiac muscle contraction. The N-terminal cardiac-specific peptide of the inhibitory subunit of troponin, cTnI (cTnI(1-39)), is a target for phosphorylation by protein kinase A (PKA) during β-adrenergic stimulation. We recently presented evidence indicating that this peptide interacts with the inhibitory peptide (cTnl(137-147)) when S23 and S24 are phosphorylated. The inhibitory peptide is also the target of the point mutation cTnI-R145G, which is associated with hypertrophic cardiomyopathy (HCM), a disease associated with sudden death in apparently healthy young adults. It has been shown that both phosphorylation and this mutation alter the cTnC-cTnI (C-I) interaction, which plays a crucial role in modulating contractile activation. However, little is known about the molecular-level events underlying this modulation. Here, we computationally investigated the effects of the cTnI-R145G mutation on the dynamics of cTn, cTnC Ca(2+) handling, and the C-I interaction. Comparisons were made with the cTnI-R145G/S23D/S24D phosphomimic mutation, which has been used both experimentally and computationally to study the cTnI N-terminal specific effects of PKA phosphorylation. Additional comparisons between the phosphomimic mutations and the real phosphorylations were made. For this purpose, we ran triplicate 150 ns molecular dynamics simulations of cTnI-R145G Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), cTnI-R145G/S23D/S24D Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), and cTnI-R145G/PS23/PS24 Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), respectively. We found that the cTnI-R145G mutation did not impact the overall dynamics of cTn, but stabilized crucial Ca(2+)-coordinating interactions. However, the phosphomimic mutations increased overall cTn fluctuations and destabilized Ca(2+) coordination. Interestingly, cTnI-R145G blunted the intrasubunit interactions between the cTnI N-terminal extension and the cTnI inhibitory peptide, which have been suggested to play a crucial role in modulating troponin function during β-adrenergic stimulation. These findings offer a molecular-level explanation for how the HCM mutation cTnI-R145G reduces the modulation of cTn by phosphorylation of S23/S24 during β-adrenergic stimulation.
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California; NSF Center for Theoretical Biological Physics, La Jolla, California. Electronic address: slindert@ucsd.edu.
authors.forename: Steffen
authors.initials: S
authors.lastname: Lindert
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington.
authors.forename: Yuanhua
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California; Department of Chemistry, University of Kentucky, Lexington, Kentucky.
authors.forename: Peter
authors.initials: P
authors.lastname: Kekenes-Huskey
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington.
authors.forename: Michael
authors.initials: M
authors.lastname: Regnier
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California; Howard Hughes Medical Institute, University of California San Diego, La Jolla, California; Department of Chemistry and Biochemistry, National Biomedical Computation Resource, University of California San Diego, La Jolla, California; NSF Center for Theoretical Biological Physics, La Jolla, California.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: McCammon
mesh_terms: D000595:Amino Acid Sequence; D006332:Cardiomegaly / Q000235:genetics*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D019210:Troponin I; D017868:Cyclic AMP-Dependent Protein Kinases