Conformational modulation of troponin T by configuration of the NH2-terminal variable region and functional effects.

Abstract

Troponin T (TnT) is an essential element in the thin filament-based regulatory system of striated muscle. Alternative mRNA splicing generates multiple TnT isoforms with primary structural differences in the NH2-terminal region. The functional significance of this hypervariable NH2-terminal domain and the developmental or muscle type-specific TnT isoforms is not fully understood. We have analyzed chicken breast muscle TnT containing a metal-binding cluster [H(E/A)EAH]4-7 (Tx) in the NH2-terminal region to demonstrate potential effects of the NH2-terminal structure on the conformation of TnT [Ogut, O., and Jin, J.-P. (1996) Biochemistry 35, 16581-16590]. Using specific antibody epitope analysis on this metal-binding TnT model, this study revealed that the binding of Zn2+ to the NH2-terminal region of chicken breast muscle TnT induces extensive conformational changes in the whole protein as demonstrated by a significant decrease in binding avidity of a polyclonal anti-TnT serum which recognizes multiple epitopes on the TnT molecule. This NH2-terminal configuration-based effect is not restricted to the metal ion interaction, whereas the binding of anti-NH2 terminus monoclonal antibodies to TnT induced similar changes. Protein-binding assays have shown that the NH2-terminal variability-induced conformational changes can alter TnT's binding affinity for tropomyosin and troponin I. The results suggest a functional modulation of TnT through the configuration of the NH2-terminal domain, and this novel mechanism may mediate the physiological significance of the TnT isoform regulation.