Energetics of the Induced Structural Change in a Ca~(2+) Regulatory Protein: Ca~(2+) and Troponin I Peptide Binding to the E41A Mutant of the N-Domain of Skeletal Troponin C

摘要

Structural studies have shown that the regulatory domains of skeletal and cardiac troponin C (sNTnC and cNTnC) undergo different conformational changes upon Ca> binding: sNTnC ~‘opens’ xvitn 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 Ca2~-bindine site I in cNTnC. Despite the striking difference, the two proteins bind their respective troponin I (TnT) regions (sTnT115..151 and cTnT14>16. respectively) in a similar open fashion. Thus, there must exist a delicate energetic balance between Ca>~ and TnT binding and the accompanying conformational changes in TnC for each system. To understand the coupling between Ca2~ and TnT binding and the concomitant structural changes, we have previously engineered an E4lA mutant of sNTnC and demonstrated that this mutation drastically reduced the Ca>-binding affinity of site Tin sNTnC, and as a result, E4IA-sNTnC remains closed in the Ca2~-bound state. Tn the present work, we investigated the interaction of E4lA-sNTnC with the sTn1115j31 peptide and found that the peptide binds to the Ca2~-saturated E4IA-sNTnC with a 1:1 stoichiometry and a dissociation constant of 300 ± 100 ~M. The peptide-induced chemical shift changes resemble those of Ca2~ binding to sNTnC, suggesting that sTnT115131 induces the “opening” of E41A-sNTnC. Tn addition, the binding of sTnT11s—131 appears to be accompanied by a conformational change in site I of E4IA-sNTnC so that the damaged regulatory site can bind Ca2~ more tightly. Without Ca2~, sTn1115131 only interacts with E4TA-sNTnC nonspecifically. When Ca2~ is titrated into E4IA-sNTnC in the presence of sTnT115.131, the Ca> binding affinity of site I was enhanced by “‘-5-fold as compared to when sTnItm>131 xvas not present. These observations suggest that the binding of Ca> and TnT is intimately coupled to each other. Together with our previous studies on Ca> and TnT 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.