Ca ²⁺ -induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy

Abstract

Muscle contraction relies on the interaction of myosin motors with F-actin, which is regulated through a translocation of tropomyosin by the troponin complex in response to Ca 2+ . The current model of muscle regulation holds that at relaxing (low-Ca 2+ ) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca 2+ ) conditions tropomyosin translocation only partially exposes myosin binding sites on F-actin so that binding of rigor myosin is required to fully activate the thin filament (TF). Here we used a single-particle approach to helical reconstruction of frozen hydrated native cardiac TFs under relaxing and activating conditions to reveal the azimuthal movement of the tropomyosin on the surface of the native cardiac TF upon Ca 2+ activation. We demonstrate that at either relaxing or activating conditions tropomyosin is not constrained in one structural state, but rather is distributed between three structural positions on the surface of the TF. We show that two of these tropomyosin positions restrain actomyosin interactions, whereas in the third position, which is significantly enhanced at high Ca 2+ , tropomyosin does not block myosin binding sites on F-actin. Our data provide a structural framework for the enhanced activation of the cardiac TF over the skeletal TF by Ca 2+ and lead to a mechanistic model for the regulation of the cardiac TF.thin filament | cardiac muscle regulation | cryoelectron microscopy

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin

“…It is noteworthy that there is a significant sterical hindrance between the myosin head and tropomyosin in "Open" position ( Fig. S3B, right), as previously shown (Risi et al 2017). Thus, these observations suggest that an "Open" tropomyosin does not fully expose the actin binding site, and a complete exposure requires a myosin binding, which displaces tropomyosin even further.…”

“…This is not the effect of glutaraldehyde fixation because the previous cryo-EM study of native/unfixed cardiac thin filament also demonstrates that ~26% of the tropomyosin segments are in "Open" position under the low calcium condition ( Fig. S4A) (Risi et al 2017). It is noteworthy that there is a significant sterical hindrance between the myosin head and tropomyosin in "Open" position ( Fig.…”

“…Note that the "helix 1" was defined as the alpha-helix that faces troponin at the level V. Because the tropomyosin colied-coil makes one turn at each actin level, the helix 1 and 2 interfere with the myosin head at the odd-numbered and the even-numbered levels, respectively. Although many of the tropomyosin segments were categorized into either "Blocked", "Closed", or "Open" positions based on the comparison with the models (PDB IDs: 5NOG, 5NOJ, and 5NOL) (Risi et al 2017), some segments were in intermediate positions between the two of the three states ("Blocked/Closed" and "Closed/Open", Table 1). At the level I, the helix 2 was in a unique position and cannot be categorized into the three states, probably because the coiled-coil structure of tropomyosin was discontinuous at the head-to-tail junction ( Fig.…”

“…We initially failed to visualize the troponin structure on native thin filaments probably due to detachment of troponin upon blotting and freezing. Thus, we fixed the filaments using 0.25% glutaraldehyde (Risi et al, 2017), which greatly improved the occupancy of troponin on the filaments (Fig. 1A).…”

“…1A). Fortunately, it has been reported that glutaraldehyde-fixation does not inhibit the calcium-dependent activation of myosin by thin filaments, although the efficiency 5 of myosin-activation and mobility of tropomyosin were slightly altered (Risi et al, 2017).…”