Crystal structure of troponin C in complex with troponin I fragment at 2.3-Å resolution

Abstract

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca 2؉ -dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca 2؉ bound state) in complex with the N-terminal fragment of TnI (TnI 1-47 ). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% (R free ‫؍‬ 32.6%). The central, connecting ␣-helix observed in the structure of uncomplexed TnC (TnC free ) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90°. As a result, TnC in the complex has a compact globular shape with direct interactions between the N-and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI 1-47 ␣-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI 1-47 ␣-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca 2؉ receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC͞TnI 1-47 structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique ␣-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca 2؉ bound conformation.

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

“…Four years later, after the publication of the crystal structure of the cTn core, this characteristic bending of TnI was confirmed by solution NMR studies. 52 A variety of studies using fluorescence, photochemical cross-linking, FRET, NMR, and crystallography provided key contact information within the cTn complex 40,[53][54][55][56][57][58][59] such that the SANS model of the ternary cTn could then be further interpreted with regards to subunit arrangements. For example, the lower lobe of the cTnC in the SANS model represents the C-terminal domain (see Figure 3).…”

“…For example, the lower lobe of the cTnC in the SANS model represents the C-terminal domain (see Figure 3). As a consequence of the antiparallel arrangement between cTnC and cTnI, 57 the lower portion of the cTnI rod contacting cTnC would be the N-domain of cTnI. In this way, the upper lobe of cTnC, which is the N-terminal domain, interacts with the regulatory region of cTnI.…”

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].…”

“…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]. The corresponding region of cTnI (cTnI ) was found to bind to the hydrophobic cleft of the C-domain of cTnC [19] and exhibit a similar conformation and orientation as observed in the structure of cTnC·3Ca 2+ ·cTnI ·cTnT 182-288 [35].…”

The Role of Troponins in Muscle Contraction

“…Therefore, the Ca 2+ -bound TnC neutralizes inhibition of the actin-activated myosin Mg 2+ -ATPase activity and initiates other interactions of TnI with TnT and Tm · actin. The C-terminal domain of TnI containing these two actin-binding sites has been proposed to act as a molecular Ca 2+ -sensitive switch during muscle contraction since it moves between Tm · actin and TnC in response to Ca 2+ binding to TnC (34,35). It has been well accepted that the metaldependent interactions of TnI and TnC play an important role (structural and functional) in the regulation of contraction.…”