The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138-12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494-28500]. The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts. Thus, while Ca2+ does not cause opening, it is required for muscle regulation. The solution structure of the cNTnC.Ca2+.cTnI147-163 complex has been determined by multinuclear multidimensional NMR spectroscopy. The structure reveals an open conformation for cNTnC, similar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helical conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presence of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B helices of cNTnC. Thus the mechanistic differences between the regulation of cardiac and skeletal muscle contraction can be understood in terms of different thermodynamics and kinetics equilibria between essentially the same structure states.

Regulation of contraction in skeletal muscle occurs through calcium binding to the protein troponin C. The solution structures of the regulatory domain of apo and calcium-loaded troponin C have been determined by multinuclear, multidimensional nuclear magnetic resonance techniques. The structural transition in the regulatory domain of troponin C on calcium binding involves an opening of the structure through large changes in interhelical angles. This leads to the increased exposure of an extensive hydrophobic patch, an event that triggers skeletal muscle contraction.

The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca 2؉ -binding properties compared with skeletal TnC and features a Ca 2؉ -binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca 2؉ -saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca 2؉ -bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca 2؉

The backbone resonance assignments have been completed for the apo ('H and "N) and calcium-loaded ('H, IsN, and 13C) regulatory N-domain of chicken skeletal troponin-C (1-90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical-shift information, along with detailed NOE analysis and 3JHNHa coupling constants, permitted the determination and quantification of the Ca2+-induced secondary structural change in the N-domain of TnC. For both structures, 5 helices and 2 short 0-strands were found, as was observed in the apo N-domain of the crystal structure of whole TnC (Herzberg 0, James MNG, 1988, JMol Biol 203:761-779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca2+ state solution structure with the apo one. The major conformational change observed is the straightening of helix-B upon Ca2+ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca2+-induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca2+ binding. The present study shows that there is virtually no change in a-helical content associated with the transition from apo to the 2Ca2+ state of the N-domain of TnC. Therefore, the Ca2+-induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.Keywords: calcium; CD; NMR; regulatory domain of troponin-C; secondary structural change Troponin-C has a key role in muscle contraction of vertebrate striated muscle (skeletal and cardiac). The binding of Ca2+ to TnC induces a conformational change that affects the interaction between TnC, troponin-I (TnI), and troponin-T (TnT). This interaction blocks the inhibitory action of TnI, allowing formation of the Mg2+-activated ATPase actomyosin complex, and ultimately leads to muscle contraction. The roles and interactions of proteins in the regulatory system of striated muscle have been studied extensively (Leavis & Gergely, 1984; Ohtsuki et al.,

The structural transition in troponin C induced by the binding of two calcium ions involves an "opening" of the structure, an event that triggers skeletal muscle contraction. We have solved the solution structure of a mutant (E41A) of the regulatory domain of skeletal troponin C wherein one bidentate ligand to the calcium in site I is missing. This structure remains "closed" upon calcium binding, indicating that the linkage between calcium binding and the induced conformational change has been broken. This provides a snapshot of skeletal troponin C between the off and on state and thereby valuable insight into the mechanism of regulation within skeletal TnC. Although several factors contribute to the triggering mechanism, the opening of the troponin C structure is ultimately dependent on one amino acid, Glu41. Insights into the structure of cardiac troponin C can also be derived from this skeletal mutant.

Cardiac troponin C (cTnC) is the Ca2؉ -dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of heart failure. Ca 2؉ is reduced ϳ 3.5-fold by bepridil and vice versa. Using multinuclear and multidimensional NMR spectroscopy, we have determined the structure of the cNTnC⅐ Ca 2؉ ⅐cTnI 147-163 ⅐bepridil ternary complex. The structure reveals a binding site for cTnI 147-163 primarily located on the A/B interhelical interface and a binding site for bepridil in the hydrophobic pocket of cNTnC⅐Ca 2؉ . In the structure, the N terminus of the peptide clashes with part of the bepridil molecule, which explains the negative cooperativity between cTnI 147-163 and bepridil for cNTnC⅐Ca 2؉ . This structure provides insights into the features that are important for the design of cTnC-specific cardiotonic drugs, which may be used to modulate the Ca 2؉ sensitivity of the myofilaments in heart muscle contraction.

Troponin is a molecular switch, directly regulating the Ca2+-dependent activation of myofilament in striated muscle contraction. Cardiac troponin is subject to covalent and noncovalent modifications; phosphorylation modulates myofilament physiology, mutations are linked to familial hypertrophic cardiomyopathy, intracellular acidification causes myocardial infarction, and cardiotonic drugs modify myofilament response to Ca2+. The structure of troponin provides insights into the mechanism of this molecular switch and an understanding of the effects of protein modification under pathophysiological conditions. Although the structure of troponin C has been solved in various Ca2+-bound states for some time, structural information on troponin I and troponin T has only emerged recently. This review summarizes recent advances on the structure of complexes of troponin subunits with the aim of assessing how these proteins interact with each other to execute its role as a molecular switch and how covalent and noncovalent modifications affect the structure of troponin and the switch mechanism. We focus on pinpointing the specific amino acid residues involved in phosphorylation and mutation and the pH sensitive regions in the structure of troponin. We also present recent structural work that have identified the docking sites of several cardiotonic drugs on cardiac troponin C and discuss their relevance in the direction of troponin based drug design in the therapy of heart disease.

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