title: Distinct conformational and functional effects of two adjacent pathogenic mutations in cardiac troponin I at the interface with troponin T.
abstract: The α-helix in troponin I (TnI) at the interface with troponin T (TnT) is a highly conserved structure. A point mutation in this region, A116G, was found in human cardiac TnI in a case of cardiomyopathy. An adjacent dominantly negative mutation found in turkey cardiac TnI (R111C, equivalent to K117C in human and K118C in mouse) decreased diastolic function and blunted beta-adrenergic response in transgenic mice. To investigate the functional importance of the TnI-TnT interface and pathological impact of the cardiac TnI mutations, we engineered K118C and A117G mutations in mouse cardiac TnI for functional studies. Despite their adjacent locations, A117G substitution results in faster mobility of cardiac TnI in SDS-PAGE whereas K118C decreases gel mobility, indicating significant and distinct changes in overall protein conformation. Consistently, monoclonal antibody epitope analysis demonstrated distinct local and remote conformational alterations in the two mutant proteins. Protein binding assays showed that K118C, but not A117G, decreased the relative binding affinity of cardiac TnI for TnT. K118C mutation decreased binding affinity for troponin C in a Ca(2+)-dependent manner, whereas A117G had a similar but less profound effect. Protein kinase A phosphorylation or truncation to remove the cardiac specific N-terminal extension of cardiac TnI resulted in similar conformational changes in the region interfacing with TnT and minimized the functional impacts of the mutations. The data demonstrate potent conformational and functional impacts of the TnT-interfacing helix in TnI and suggest a role of the N-terminal extension of cardiac TnI in modulating TnI-TnT interface functions.
authors: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, United States.
authors: Shirin
authors: S
authors: Akhter
authors: Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201, United States.
authors: J-P
authors: JP
authors: Jin
keywords: BSA, bovine serum albumin; Cardiac muscle; ELISA, enzyme linked immunosorbent assay; McTnI, wild type mouse cardiac TnI; McTnI-ND, N-terminal truncated mouse cardiac TnI; McTnI-NDA117G, N-terminal truncated McTnIA117G; McTnI-NDK118C, N-terminal truncated McTnIK118C; McTnIA117G, mouse cardiac TnI A117G mutation; McTnIK118C, mouse cardiac TnI K118C mutation; PKA, protein kinase A; Protein conformation; TnC, troponin C; TnI, troponin I; TnT, troponin T; Troponin; Troponin I mutation; Troponin I–Troponin T interface