Switch action of troponin on muscle thin filament as revealed by spin labeling and pulsed EPR

Abstract

We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to measure the distance between spin labels at Cys(133) of the regulatory region of TnI (TnI133) and a native or genetically substituted cysteine of TnC (TnC44, TnC61, or TnC98). In the +Ca(2+) state, the TnC44-TnI133-T distance was 42 A, with a narrow distribution (half-width of 9 A), suggesting that the regulatory region binds the N-lobe of TnC. Distances for TnC61-TnI133 and TnC98-TnI133 were also determined to be 38 A (width of 12 A) and 22 A (width of 3.4 A), respectively. These values were all consistent with recently published crystal structure (Vinogradova, M. V., Stone, D. B., Malanina, G. G., Karatzaferi, C., Cooke, R., Mendelson, R. A., and Fletterick, R. J. (2005) Proc. Natl Acad. Sci. U.S.A. 102, 5038-5043). Similar distances were obtained with the same spin pairs on a reconstituted thin filament in the +Ca(2+) state. In the -Ca(2+) state, the distances displayed broad distributions, suggesting that the regulatory region of TnI was physically released from the N-lobe of TnC and consequently fluctuated over a variety of distances on a large scale (20-80 A). The interspin distance appeared longer on the filament than on troponin alone, consistent with the ability of the region to bind actin. These results support a concept that the regulatory region of TnI, as a molecular switch, binds to the exposed hydrophobic patch of TnC and traps the inhibitory region of TnI away from actin in Ca(2+) activation of muscle.

FIGURE 1.

Analysis of the PELDOR spectra of doubly labeled TnC and TnI in the reconstituted heterotrimer complex. Spectra were taken with a four-pulse sequence, both in the presence (red line) and absence (blue line) of Ca²⁺ ions at 80 K. Spin pairs were analyzed between Cys¹³³ in TnI and three different residues in TnC: Cys⁴⁴ (a), Cys⁶¹ (b), and Cys⁹⁸ (c). For each pair, the left panel compares the experimental spectrum modulation of the doubly labeled complex (dotted line) with the fitted spectrum modulation (continuous line). The single or double Gaussian distribution shown in the right panel fits the experimental spectra satisfactorily. In the right panel of a, a second population (56%) of widely separated noninteracting spins (>80 Å) in addition to the single Gaussian distribution is needed (not shown) to obtain the best fit for the complex in the −Ca²⁺ state (blue line).

FIGURE 2.

Analysis of the PELDOR spectra of interacting doubly labeled troponin C and I in the reconstituted thin filament. The spectra were taken in the presence (red line) and absence (blue line) of Ca²⁺ ions and are shown as described in Fig. 1. Spin pairs were between Cys¹³³ in TnI and two different residues in TnC: Cys⁴⁴ (a) and Cys⁶¹ (b).

FIGURE 3.

Proposed conformations of TnC and TnI in the heterotrimer complex (a) and in the thin filament (b). a, in the presence of Ca²⁺ ions, the switch segment adjacent to Cys¹³³ of TnI is attached to N-lobe of TnC, which is in the open state in the presence of Ca²⁺ ions as in the crystal structure. In the absence of Ca²⁺ ions, the switch segment is dissociated from the N-lobe, which is in the closed state and remains mobile. b, the same bond is made in the presence of Ca²⁺ ions, whereas the C-terminal region proximal to Cys¹³³ may interact with actin in the absence of Ca²⁺ ions. The structures of the ternary TnC-TnI-TnT2 complexes in the presence and absence of Ca²⁺ ions are modified from the PDB codes 1YTZ and 1YVO, respectively. Only one of the two troponin complexes is shown, drawn with an α-carbon ribbon model. TnC is in purple, TnI is in light blue, and TnT is in dark yellow. The residues Gly⁴⁴, Ala⁶¹, and Glu⁹⁸ of chicken TnC and Asn¹³³ of chicken TnI are shown with a space-filling model. The C-terminal extension (a putative second actin-binding domain) of TnI and N-terminal extensions of TnT (TnT1) are drawn schematically as ellipsoids. The shaded regions indicate two putative TnI-actin interactions in the thin filament in the −Ca²⁺ state. Tropomyosin strands and six actin protomers are drawn as brown sticks and light blue spheres, respectively.