Energetics of the induced structural change in a Ca2+ regulatory protein: Ca2+ and troponin I peptide binding to the E41A mutant of the N-domain of skeletal troponin C

Structural studies have shown that the regulatory domains of skeletal and c ardiac troponin C (sNTnC and cNTnC) undergo different conformational change s upon Ca2+ binding; sNTnC "opens" with a large exposure of the hydrophobic surface, while cNTnC retains a "closed" conformation similar to that in th e apo state. This is mainly due to the fact that there is a defunct Ca2+-bi nding site I in cNTnC. Despite the striking difference, the two proteins bi nd their respective troponin I (TnI) regions (sTnI(115-131) and CTnI(147-16 3), respectively) in a similar open fashion. Thus, there must exist a delic ate energetic balance between Ca2+ and TnI binding and the accompanying con formational changes in TnC for each system. To understand the coupling betw een Ca2+ and TnI binding and the concomitant structural changes, we have pr eviously engineered an E41A mutant of sNTnC and demonstrated that this muta tion drastically reduced the Ca2+-binding affinity of site I in sNTnC, and as a result, E41A-sNTnC remains closed in the Ca2+-bound state. In the pres ent work, we investigated the interaction of E41A-sNTnC with the sTnI(115-1 31) peptide and found that the peptide binds to the Ca2+-saturated E41A-sNT nC with a 1:1 stoichiometry and a dissociation constant of 300 +/- 100 muM. The peptide-induced chemical shift changes resemble those of Ca2+ binding to sNTnC, suggesting that sTnI(115-131) induces the "opening" of E41A-sNTnC . In addition, the binding of sTnI(115-131) appears to be accompanied by a conformational change in site I of E41A-sNTnC so that the damaged regulator y site can bind Ca2+ more tightly. Without Ca2+, sTnI(115-131) only interac ts with E41A-sNTnC nonspecifically. When Ca2+ is titrated into E41A-sNTnC i n the presence of sTnI(115-131), the Ca2+-binding affinity of site I was en hanced by similar to5-fold as compared to when sTnI(115-131) was not presen t. These observations suggest that the binding of Ca2+ and TnI is intimatel y coupled to each other. Together with our previous studies on Ca2+ and TnI peptide binding to sNTnC and cNTnC, these results allow us to dissect the mechanism and energetics of coupling of ligand binding and structural openi ng intricately involved in the regulation of skeletal and cardiac muscle co ntraction.