Localization of The Tropomyosin-Binding Sites in Troponin T And Functional Suppression of An Error Splicing of Its C-Terminal Variable Region

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The interaction between troponin T (TnT) and tropomyosin (Tm) is pivotal in the Ca2+-regulation of muscle contraction. It has been known for three decades that TnT has two binding sites for Tm. The conserved middle and the C-terminal regions of TnT each contain a Tm-binding site and both sites are critical for the function of muscle thin filament. However, the precise locations of the Tm-binding sites have not been identified. By mAb competition assays, we located the middle region Tm-binding site of TnT in the beginning of the conserved sequence. Previous data showed that deletion of the C-terminal 14 amino acids in TnT did not reduce Tm-binding. Cardiac TnT with a longer deletion of the C-terminal 28 amino acids also retained the C-terminal Tm-binding site as shown by its dominant cardiomyopathy phenotype that indicates effective myofilament incorporation. In contrast, a truncation of slow TnT deleting the C-terminal 83 amino acids due to a nonsense mutation significantly lowered Tm-binding affinity and causes a recessive form of nemaline myopathy. Considering the known crystal structure of partial troponin, we further tested additional deletions to locate the C-terminal region Tm-binding site of TnT in the beginning of the T2 segment. Different from the dominant C-terminal truncation mutation of cardiac TnT, an error-splicing of the mutually exclusive exon 16 and exon 17 in fast skeletal muscle TnT significantly lowered Tm-binding affinity by deleting the C-terminal 28 amino acids and replacing it with a long non-sense peptide. The suppression of potentially harmful effects of the splicing error provides a mechanism to protect muscle function.