NMR Studies of Ca 2+ 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 † (original) (raw)
Ca 2+ binding to the N-domain of skeletal muscle troponin C (sNTnC) induces an "opening" of the structure Nat. Struct. Biol. 2,[784][785][786][787][788][789], which is typical of Ca 2+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 Ca 2+ -saturated states. Both of these proteins are modified in Ca 2+ -binding site I, albeit differently, suggesting a critical role for this region in the coupling of Ca 2+ binding to the induced structural change. To understand the mechanism and the energetics involved in the Ca 2+ -induced structural transition, Ca 2+ binding to E41A sNTnC and to cNTnC have been investigated by using one-dimensional 1 H and two-dimensional { 1 H, 15 N}-HSQC NMR spectroscopy. Monitoring the chemical shift changes during Ca 2+ 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 Ca 2+ binding affinity of the site I by ∼100-fold [from K D2 ) 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 K D1 ) 1.7 µM (sNTnC) to 15 µM (E41A sNTnC)]. Ca 2+ titration of cNTnC confirms that cNTnC binds only one Ca 2+ with a determined dissociation constant K D of 2.6 µM. The Ca 2+ -induced chemical shift changes occur over the entire sequence in cNTnC, suggesting that the defunct site I is perturbed when site II binds Ca 2+ . These measurements allow us to dissect the mechanism and energetics of the Ca 2+ -induced structural changes. Abstract published in AdVance ACS Abstracts, September 15, 1997. 1 Abbreviations: TnC, troponin C; sTnC, skeletal troponin C; cTnC, cardiac troponin C; cNTnC, N-domain (residues 1-89) of recombinant human cardiac troponin C; sNTnC, N-domain (residues 1-90) of recombinant chicken skeletal troponin C; NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum coherence; NOE, nuclear Overhauser effect; KD1, dissociation constant of the first Ca 2+ binding; KD2, dissociation constant of the second Ca 2+ binding.