Structure and Dynamics of the C-domain of Human Cardiac Troponin C in Complex with the Inhibitory Region of Human Cardiac Troponin I

Abstract

Cardiac troponin C is the Ca 2؉ -dependent switch for heart muscle contraction. Troponin C is associated with various other proteins including troponin I and troponin T. The interaction between the subunits within the troponin complex is of critical importance in understanding contractility. Following a Ca 2؉ signal to begin contraction, the inhibitory region of troponin I comprising residues Thr 128 -Arg 147 relocates from its binding surface on actin to troponin C, triggering movement of troponin-tropomyosin within the thin filament and thereby freeing actin-binding site(s) for interactions with the myosin ATPase of the thick filament to generate the power stroke. The structure of calcium-saturated cardiac troponin C (C-domain) in complex with the inhibitory region of troponin I was determined using multinuclear and multidimensional nuclear magnetic resonance spectroscopy. The structure of this complex reveals that the inhibitory region adopts a helical conformation spanning residues Leu 134 -Lys 139 , with a novel orientation between the E-and H-helices of troponin C, which is largely stabilized by electrostatic interactions. By using isotope labeling, we have studied the dynamics of the protein and peptide in the binary complex. The structure of this inhibited complex provides a framework for understanding into interactions within the troponin complex upon heart contraction.The binding of Ca 2ϩ to the troponin complex initiates cardiac muscle contraction (1-5). The troponin complex is composed of three subunits: troponin C (TnC), 1 troponin I (TnI), and troponin T (TnT) (6). The three subunits of the troponin complex are necessary for Ca 2ϩ -induced regulation of cardiac muscle contractility. TnC, the Ca 2ϩ -sensitive component of the complex, is a member of the EF-hand family of Ca 2ϩ -binding proteins and contains two high affinity Ca 2ϩ /Mg 2ϩ -binding sites (sites III and IV) in the C-terminal domain and one low affinity Ca 2ϩ -binding site (site II) in the N-terminal domain (7-9). At physiological conditions during muscle relaxation, the two C-terminal Ca 2ϩ /Mg 2ϩ -binding sites are occupied, and the N-terminal Ca 2ϩ -binding site is unoccupied. During the onset of muscle contraction, a transient increase in cytosolic Ca 2ϩ concentrations allows the low affinity N-terminal domain to bind Ca 2ϩ , resulting in the initiation of heart contraction (10). TnI is the subunit that in the presence of tropomyosin inhibits myosin Mg 2ϩ -ATPase activity. TnI inhibition is removed by the binding of Ca 2ϩ to the N-terminal domain of the TnC subunit (11). TnT is the subunit that binds tropomyosin, TnC, and TnI, anchoring the troponin complex to the thin filament and aids in the propagation of Ca 2ϩ -induced conformational changes (12, 13).The structure of cardiac TnC, sharing sequence and structural similarities to the skeletal isoform, has been solved by both x-ray crystallography and NMR spectroscopy (7,(15)(16)(17)(18)(19). Upon Ca 2ϩ binding, there is an opening of the N-domain of the skeletal isoform, whereas th...

Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex

“…Many recent studies have tended to focus on the interactions between the domains of TnC and peptides from TnI and TnT and have mapped out the interacting regions in some detail. Structures of complexes between TnC domains and TnI peptides (13)(14)(15)(16)(17) have revealed the nature of these interactions at atomic detail and have been confirmed by the recent x-ray structures of cardiac (5) and skeletal troponin (18).…”

Sarcomeric Proteins and Familial Hypertrophic Cardiomyopathy: Linking Mutations in Structural Proteins to Complex Cardiovascular Phenotypes

“…Some of the lingering questions regarding the mechanism of the regulation of Ca 2+ dependent myofilament activation by the thin filament have been clarified by the development of a series of high resolution structures of the binary TnC:TnI complex [53,54]. These studies were supported and extended by the landmark publication of the Ca 2+ -activated core domain structure of human cardiac troponin (HcTn) in 2003 [55].…”

Interaction of cardiac troponin with cardiotonic drugs: A structural perspective

“…NMR studies of TnC with various TnI peptides have yielded detailed structural information on the structure of TnC when bound to TnI [16][17][18][19], on the structure of TnI inhibitory peptide [20,21], and on the overall topology of TnC-TnI arrangement [22][23][24][25][26][27][28][29][30][31][32]. The high-resolution structures of TnC-TnI available are the X-ray structure of sTnC·2Ca 2+ ·sTnI [33], the NMR structures of cNTnC·Ca 2+ ·cTnI 147-163 [13], sNTnC(rhodamine)·2Ca 2+ ·sTnI 115-131 [14], and cCTnC·2Ca 2+ ·cTnI 128-147 [34], the X-ray structure of the core domain cardiac troponin complex, cTnC·3Ca 2+ ·cTnI ·cTnT2 182-288 [35], and the X-ray structures of skeletal troponin complex in both the apo and Ca 2+ -state, sTnC·apo·sTnI ·sTnT 156-262 and sTnC·4Ca 2+ ·sTnI ·sTnT [36]. In the structure of sTnC·2Ca 2+ ·sTnI , the 31-residue long sTnI α-helix (residues 3-33) stretches on the surface of the sTnC and stabilizes its compact conformation by multiple contacts with both TnC domains [33].…”