NMR Studies of Ca2+ Binding to the Regulatory Domains of Cardiac and E41A Skeletal Muscle Troponin C Reveal the Importance of Site I to Energetics of the Induced Structural Changes

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Abstract
Ca2+ binding to the N-domain of skeletal muscle troponin C (sNTnC) induces an “opening” of the structure [Gagné, S. M., et al. (1995) Nat. Struct. Biol. 2, 784−789], which is typical of Ca2+-regulatory proteins. However, the recent structures of the E41A mutant of skeletal troponin C (E41A sNTnC) [Gagné, S. M., et al. (1997) Biochemistry 36, 4386−4392] and of cardiac muscle troponin C (cNTnC) [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216−18221] reveal that both of these proteins remain essentially in the “closed” conformation in their Ca2+-saturated states. Both of these proteins are modified in Ca2+-binding site I, albeit differently, suggesting a critical role for this region in the coupling of Ca2+ binding to the induced structural change. To understand the mechanism and the energetics involved in the Ca2+-induced structural transition, Ca2+ binding to E41A sNTnC and to cNTnC have been investigated by using one-dimensional 1H and two-dimensional {1H,15N}-HSQC NMR spectroscopy. Monitoring the chemical shift changes during Ca2+ titration of E41A sNTnC permits us to assign the order of stepwise binding as site II followed by site I and reveals that the mutation reduced the Ca2+ binding affinity of the site I by ∼100-fold [from KD2 = 16 μM [sNTnC; Li, M. X., et al. (1995) Biochemistry 34, 8330−8340] to 1.3 mM (E41A sNTnC)] and of the site II by ∼10-fold [from KD1 = 1.7 μM (sNTnC) to 15 μM (E41A sNTnC)]. Ca2+ titration of cNTnC confirms that cNTnC binds only one Ca2+ with a determined dissociation constant KD of 2.6 μM. The Ca2+-induced chemical shift changes occur over the entire sequence in cNTnC, suggesting that the defunct site I is perturbed when site II binds Ca2+. These measurements allow us to dissect the mechanism and energetics of the Ca2+-induced structural changes.