The N-Terminal Hypervariable Region of Troponin T Differentially Modulates the Affinity of Tropomyosin-Binding Sites

Abstract

The troponin complex plays a central role in the allosteric function of sarcomeric thin filaments by enacting conformational changes during the Ca2+-regulated contraction and relaxation of striated muscle. The troponin subunit T (TnT) has two binding sites for tropomyosin (Tm) and is responsible for anchoring the troponin complex to the thin filament. Although the C-terminal and middle regions of the TnT polypeptide chain are highly conserved among the three muscle type isoforms, the hypervariable N-terminal region has evolutionarily diverged significantly among isoforms. Previous studies have shown that the N-terminal variable region fine-tunes Ca2+ regulation of muscle contractility via modulation of the overall molecular conformation of TnT, and its interactions with Tm. In the present study, we engineered intact TnT and representative fragments of TnT, expressed them in E. coli, and prepared purified proteins for functional studies. Tropomyosin binding affinity was analyzed using affinity chromatography and solid phase protein binding assays to investigate the modulatory effects of the N-terminal variable region. The results demonstrated that in the absence of the N-terminal variable region, TnT's conserved middle region and C-terminal T2 region Tm-binding sites showed comparable Tm-binding affinities across isoforms. The data demonstrate that without the modulatory effect of the N-terminal variable region, the intrinsic Tm-binding affinities of the two sites are both high. In contrast, the presence of the isoform specific N-terminal variable region differentially reduces the binding affinity of TnT for Tm, primarily at the middle region binding site. These novel findings indicate that the N-terminal variable region plays a key role in the functional difference of muscle fiber type-specific, developmental, splice variant, and pathogenic TnT isoforms by modulating the interactions with Tm during the contraction and relaxation of cardiac and skeletal muscle.