Conformational modulation of slow skeletal muscle troponin T by an NH2-terminal metal-binding extension

Troponin T (TnT) is an essential element in the thin filament Ca2+-regulato ry system controlling striated muscle contraction. Alternative RNA splicing generates developmental and muscle type-specific TnT isoforms differing in the hypervariable NH2-terminal region. Using avian fast skeletal muscle Tn T containing a metal-binding segment, we have demonstrated a role of the NH 2-terminal domain in modulating the conformation of TnT (Wang J and Jin JP. Biochemistry 37: 14519-14528, 1998). To further investigate the structure- function relationship of TnT, the present study constructed and characteriz ed a recombinant protein in which the metal-binding peptide present in avia n fast skeletal muscle TnT was fused to the NH2 terminus of mouse slow skel etal muscle TnT. Metal ion or monoclonal antibody binding to the NH2-termin al extension induced conformational changes in other domains of the model T nT molecule. This was shown by the altered affinity to a monoclonal antibod y against the COOH-terminal region and a polyclonal antiserum recognizing m ultiple epitopes. Protein binding assays showed that metal binding to the N H2-terminal extension had effects on the interaction of TnT with troponin I , troponin C, and most significantly, tropomyosin. The data indicate that t he NH2-terminal Tx [4-7 repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-H is] extension confers a specific conformational modulation in the slow skel etal muscle TnT.