Ca2+-induced conformational transition in the inhibitory and regulatory regions of cardiac troponin I

Abstract

Cardiac muscle activation is initiated by the binding of Ca2+ to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca2+ binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using Förster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg2+-saturated) and Ca2+-activated (Ca2+-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca2+. Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca2+-induced increases of >8 Å. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca2+ activation causes residues within cTnI-I to switch from a β-turn/coil to an extended quasi-α-helical conformation as the actin-contacts are broken, whereas cTnI-R remains α-helical in both Mg2+- and Ca2+-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg2+- and Ca2+-saturated states.