3D-Reconstruction Reveals the Organization of Troponin on Cardiac Thin Filaments

Abstract

Muscle contraction is activated through Ca2+-binding to TnC, leading to tropomyosin movement and unblocking of myosin binding sites on actin. To elucidate this regulatory mechanism, the 3D organization of the regulatory proteins on the thin filament must be determined. While tropomyosin arrangement has been determined by 3DEM using helical reconstruction and IHRSR analysis, troponin was not visualized due to helical averaging. To solve the structure of native cardiac thin filaments in low-Ca2+, we have used single particle reconstruction of negatively stained specimens, without imposing actin helical symmetry. To acquire identical particles for reconstruction, a cross-correlation approach was applied to determine the axial position of troponin. The reconstruction had a resolution of 2.5 nm. Nine different reference models were used to test for any model bias in the reconstruction. Eight were variations of an earlier model (Pirani et al., JMB, 2006), with troponin translated ±2 nm along or ±20oazimuthally around the filament, or tilted ±15oradially or azimuthally; one model used a sphere to represent troponin. With eight of the models, the reconstruction converged to the same final structure, which clearly showed F-actin, tropomyosin and troponin densities. Consistency between reconstructions from multiple different models indicates that the troponin densities are reliable. Atomic models of tropomyosin and actin fitted well into the reconstruction. The densities attributable to troponin were also well fitted by the cardiac troponin core domain (Takeda et al., Nature 2003), with the Tn “IT” arm projecting at ∼50o to the filament axis (cf. Knowles et al., JMB 2012). The core domain orientation and the TnT tail, observed as a widening of tropomyosin toward the barbed end of filaments, established the polarity of troponin on the thin filament. Our 3DEM also is consistent with cross-linking data localizing troponin on actin-tropomyosin.