Troponin-I interacts with the met47 region of skeletal muscle actin. implications for the mechanism of thin filament regulation by calcium

Striated muscles are regulated by Ca2+via the thin filament proteins troponin (Tn) and tropomyosin (Tm). In the absence of Ca2+, contraction is inhibited, whereas myosin-actin interaction and contraction can take place in its presence. Although it is well established that the interaction of troponin-I (TnI), the inhibitory subunit of Tn, with actin is required for the inhibition process and that there are two separate actin-binding regions in TnI that interact with actin, the molecular mechanism of this inhibition process is still not clear. Using TnI mutants with photocrosslinking probes attached to genetically engineered cysteine residues in each of the two actin-binding regions, we show that both regions are close to Met47 of actin in its outer domain. It has been proposed that the Ca2+-induced activation of contraction involves the movement of Tm from the outer to the inner domain of the actin filament. On the basis of our results presented here, we propose that the position of Tm at the outer domain of actin in the Ca2+-free state is stabilized by the presence of TnI over actin’s outer domain via mutual interactions of all three components. In the presence of Ca2+, TnI’s actin-binding regions dissociate from actin allowing Tm to move toward actin’s inner domain.