Structure of cardiac muscle troponin C unexpectedly reveals a closed regulatory domain.

Samuel K Sia,Brian D Sykes,Stéphane M Gagné,Leo Spyracopoulos,Wen Liu,John A Putkey,Monica X Li

doi:10.1074/jbc.272.29.18216

Abstract

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 Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca2+-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 Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction.

Highlights

Transient increases in cytosolic Ca2ϩ levels in the cardiac muscle cell must be recognized by the thin filament to regulate cardiac muscle contraction
We show that, in contrast to predicted models [7,8,9], the analogous conformational change does not occur in cardiac troponin C (TnC), and that this is the direct structural consequence of inactivating Ca2ϩ-binding site I
It has been shown that the conversion of these Cys residues to Ser residues has no effect on the ability of cardiac TnC to recover ATPase activity in TnC-extracted fast skeletal and cardiac myofibrils, and has little effect on Ca2ϩ binding to site II of cardiac TnC [11]

Summary

Introduction

Transient increases in cytosolic Ca2ϩ levels in the cardiac muscle cell must be recognized by the thin filament to regulate cardiac muscle contraction This critical function is accomplished by cardiac TnC1 (161 residues), a member of the EFhand family of Ca2ϩ-binding proteins, which relays the Ca2ϩ signal via a conformational change to the rest of the troponintropomyosin complex, and signals the activation of the myosin-actin ATPase reaction. For the purposes of this study, the two Cys residues at positions 35 and 84 of wild type cardiac TnC have been mutated to Ser residues This prevents the formation of intra- and intermolecular disulfide bonds, which confer Ca2ϩ-independent activity to cardiac TnC when assayed in skeletal muscle myofibrils [11]. Structural statistics for the 30 lowest energy structures (Table I) show that the N- and Cdomains are very well defined separately, with the central linker shown to be flexible by relaxation measurements.

Methods

Results

Conclusion