Energetics of the Induced Structural Change in a Ca<sup>2+</sup> Regulatory Protein:  Ca<sup>2+</sup> and Troponin I Peptide Binding to the E41A Mutant of the N-Domain of Skeletal Troponin C<sup>†</sup>

Structural studies have shown that the regulatory domains of skeletal and cardiac troponin C (sNTnC and cNTnC) undergo different conformational changes upon Ca<sup>2+</sup> binding; sNTnC “opens” with a large exposure of the hydrophobic surface, while cNTnC retains a “closed” conformation similar to that in the apo state. This is mainly due to the fact that there is a defunct Ca<sup>2+</sup>-binding site I in cNTnC. Despite the striking difference, the two proteins bind their respective troponin I (TnI) regions (sTnI<sub>115</sub><sub>-</sub><sub>131</sub> and cTnI<sub>147</sub><sub>-</sub><sub>163</sub>, respectively) in a similar open fashion. Thus, there must exist a delicate energetic balance between Ca<sup>2+</sup> and TnI binding and the accompanying conformational changes in TnC for each system. To understand the coupling between Ca<sup>2+</sup> and TnI binding and the concomitant structural changes, we have previously engineered an E41A mutant of sNTnC and demonstrated that this mutation drastically reduced the Ca<sup>2+</sup>-binding affinity of site I in sNTnC, and as a result, E41A-sNTnC remains closed in the Ca<sup>2+</sup>-bound state. In the present work, we investigated the interaction of E41A-sNTnC with the sTnI<sub>115</sub><sub>-</sub><sub>131</sub> peptide and found that the peptide binds to the Ca<sup>2+</sup>-saturated E41A-sNTnC with a 1:1 stoichiometry and a dissociation constant of 300 ± 100 μM. The peptide-induced chemical shift changes resemble those of Ca<sup>2+</sup> binding to sNTnC, suggesting that sTnI<sub>115</sub><sub>-</sub><sub>131</sub> induces the “opening” of E41A-sNTnC. In addition, the binding of sTnI<sub>115</sub><sub>-</sub><sub>131</sub> appears to be accompanied by a conformational change in site I of E41A-sNTnC so that the damaged regulatory site can bind Ca<sup>2+</sup> more tightly. Without Ca<sup>2+</sup>, sTnI<sub>115</sub><sub>-</sub><sub>131</sub> only interacts with E41A-sNTnC nonspecifically. When Ca<sup>2+</sup> is titrated into E41A-sNTnC in the presence of sTnI<sub>115</sub><sub>-</sub><sub>131</sub>, the Ca<sup>2+</sup>-binding affinity of site I was enhanced by ∼5-fold as compared to when sTnI<sub>115</sub><sub>-</sub><sub>131</sub> was not present. These observations suggest that the binding of Ca<sup>2+</sup> and TnI is intimately coupled to each other. Together with our previous studies on Ca<sup>2+</sup> 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 opening intricately involved in the regulation of skeletal and cardiac muscle contraction.