Kinetic analysis of the interactions between Troponin C (TnC) and Troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca2+/Mg2+‐dependent interactions between TnC and TnI play a critical role in regulating the ‘on’ and ‘off’ states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N‐terminus of TnI (residues 1–40 of skeletal TnI) and skeletal TnC in the presence of Ca2+ ions, Mg2+ ions and in the presence of the C‐terminal regulatory region peptides: TnI96–115, TnI96–131 and TnI96–139. Our results show the N‐terminus of TnI can bind to TnC with high affinity in the presence of Ca2+ or Mg2+ ions with apparent equilibrium dissociation constants of K d Ca 2+ = 48 nM and K d Mg 2+ = 29 nM. The apparent association and dissociation rate constants for the interactions were, kon = 4.8 × 105 M −1 s−1, 3.4 × 105 M −1 s−1 and koff = 2.3 × 10−2 s−1, 1.0 × 10−2 s−1 for TnC(Ca2+) and TnC(Mg2+) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI96–139 (or TnI96–131) and TnC–TnI1–40. Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI96–115 and TnI116–131) and the presence of Ca2+. Furthermore, the presence of TnI1–40 slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI–TnC interaction where the N‐terminus of TnI remains bound to the C‐domain of TnC in the presence of high and low Ca2+ levels while the TnI regulatory region (residues 96–139) switches in its binding interactions between the actin‐tropomyosin thin filament and its own sites on the N‐ and C‐domain of TnC at high Ca2+ levels, thus regulating muscle contraction. Copyright © 2003 John Wiley & Sons, Ltd.