A Kinetic Model for the Binding of Ca2+to the Regulatory Site of Troponin from Cardiac Muscle

Wen-Ji Dong; Chien-Kao Wang; Albert M Gordon; Steven S Rosenfeld; Herbert C Cheung

doi:10.1074/jbc.272.31.19229

The kinetics of the binding of Ca 21 to the single regulatory site of cardiac muscle troponin was investigated by using troponin reconstituted from the three sub-units, using a monocysteine mutant of troponin C (cTnC) labeled with the fluorescent probe 2-[(4*-(iodoac-etamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-35. The kinetic tracings of binding experiments for troponin determined at free [Ca 21 ] > 1 mM were resolved into two phases. The rate of the fast phase increased with increasing [Ca 21 ], reaching a maximum of about 35 s 21 at 4 °C, and the rate of the slow phase was approximately 5 s 21 and did not depend on [Ca 21 ]. Dissociation of bound Ca 21 occurred in two phases, with rates of about 23 and 4 s 21. The binding and dissociation results obtained with the binary complex formed between cardiac troponin I and the IAANS-labeled cTnC mutant were very similar to those obtained from reconstituted troponin. The kinetic data are consistent with a three-step sequential model similar to the previously reported mechanism for the binding of Ca 21 to a cTnC mutant labeled with the same probe at Cys-84 (Dong et al. (1996) J. Biol. Chem. 271, 688 – 694). In this model, the initial binding in the bimolecular step to form the Ca 21-tropo-nin complex is assumed to be a rapid equilibrium, followed by two sequential first-order transitions. The apparent bimolecular rate constant is 5.1 3 10 7 M 21 s 21 , a factor of 3 smaller than that for cTnC. The rates of the first-order transitions are an order of magnitude smaller for troponin than for cTnC. These kinetic differences form a basis for the enhanced Ca 21 affinity of troponin relative to the Ca 21 affinity of isolated cTnC. Phosphorylation of the monocysteine mutant of tropo-nin I by protein kinase A resulted in a 3-fold decrease in the bimolecular rate constant but a 2-fold increase in the two observed Ca 21 dissociation rates. These changes in the kinetic parameters are responsible for a 5-fold reduction in Ca 21 affinity of phosphorylated troponin for the specific site. Muscle contraction consists of a cascade of events involving several protein structural changes and protein-protein interactions within the thick and thin filaments (1). For contraction to occur, the N-terminal domain of the myosin heavy chain in the thick filament must first bind to actin in the thin filament. The formation of this active actomyosin complex is, however, inhibited by troponin I. This inhibitory action is regulated in vertebrate skeletal and cardiac muscle through the binding of calcium to another troponin subunit, troponin C. This Ca 21 binding releases the inhibition of formation of the actomyosin complex. The crystal structures of sTnC 1 from turkey (2, 3) and chicken (4, 5) reveal a dumbbell-shaped molecule with two globular domains connected by a long central helix. Each domain contains two metal ion binding sites, designated as sites I and II in the N-domain and sites III and IV in the C-domain. Sites III and IV have a relatively high affinity for Ca 21 (K a ' 10 7 M 21) and also bind Mg 21 competitively (K a ' 10 3 M 21), and sites I and II have a lower Ca 21 affinity and are specific for Ca 21 (K a ' 10 5 M 21). Current evidence indicates that the two Ca/Mg sites in the C-domain most likely play a structural role, and the Ca 21-specific sites in the N-domain carry out a regulatory function. Cardiac muscle troponin C differs from the skeletal muscle isoform in that site I cannot bind Ca 21 due to several amino acid substitutions in critical positions within the 12-residue Ca 21-binding loop. Although the three-dimensional structure of cTnC has not been determined, it is reasonable to assume, on the basis of sequence homology and similarities in physiologic functions, that its structure is similar to that of sTnC. Reversible Ca 21 binding to the single Ca 21-specific site is believed to induce conformational changes in the N-domain, and these changes appear to modulate the troponin C-troponin I interaction. The precise nature of these conformational changes are still obscure. A useful biophysical approach to delineate Ca 21-induced global structural changes is the use of extrinsic fluorescent probes that are attached to TnC. cTnC contains two cysteine residues, Cys-35 located in the nonfunctional Ca 21-binding loop I and Cys-84 located in the C-terminal end of helix D where the cTnC-cTnI interaction occurs. In a previous study of two monocysteine mutants of cTnC (6), we showed that the probe IAANS covalently linked to Cys-84 is partially buried and sensitive to Ca 21 binding to the single Ca 21-specific site. The same probe attached to Cys-35, however, is highly exposed to solvent and not sensitive to Ca 21 binding. Once incorporated into troponin, the probe attached to the two cysteine residues have very different properties. The fluorescence of IAANS attached to Cys-84 of cTnC within the troponin complex is insensitive to Ca 21 binding, but the fluorescence of the probe attached to Cys-35 within the complex decreases by a factor of 3

A Kinetic Model for the Binding of Ca2+to the Regulatory Site of Troponin from Cardiac Muscle

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