title: 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.
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 approximately 100-fold [from KD2 = 16 microM [sNTnC; Li, M. X., et al. (1995) Biochemistry 34, 8330-8340] to 1.3 mM (E41A sNTnC)] and of the site II by approximately 10-fold [from KD1 = 1.7 microM (sNTnC) to 15 microM (E41A sNTnC)]. Ca2+ titration of cNTnC confirms that cNTnC binds only one Ca2+ with a determined dissociation constant KD of 2.6 microM. 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.
authors.affiliation: MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: M X
authors.initials: MX
authors.lastname: Li
authors.forename: S M
authors.initials: SM
authors.lastname: Gagné
authors.forename: L
authors.initials: L
authors.lastname: Spyracopoulos
authors.forename: C P
authors.initials: CP
authors.lastname: Kloks
authors.forename: G
authors.initials: G
authors.lastname: Audette
authors.forename: M
authors.initials: M
authors.lastname: Chandra
authors.forename: R J
authors.initials: RJ
authors.lastname: Solaro
authors.forename: L B
authors.initials: LB
authors.lastname: Smillie
authors.forename: B D
authors.initials: BD
authors.lastname: Sykes
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004734:Energy Metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism*
chemical_list: D019209:Troponin C; D002118:Calcium