High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex

Abstract

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8 Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

Keywords: actin; cardiac muscle; cryoelectron microscopy; myosin.

Fig. 1. Intermolecular interactions in the cardiac actomyosin rigor complex.

(A) Atomic model of the cardiac actomyosin complex shows structural elements of cardiac myosin involved in interactions with actin and tropomyosin: CM loop (blue ribbons), loop 2 (red ribbons), loop 4 (yellow ribbons), HLH motif (cyan ribbons), and loop 3 (brown ribbons). Actin atoms are tan, tropomyosin is black, while myosin motor domain is plum. Myosin residues involved in interactions with actin are in magenta while myosin residues involved in intra-myosin contacts are in green. (B-H) Detailed interfaces between myosin tropomyosin and actin are shown for CM loop (B and C), loop 2 (D), loop 4 (E), HLH motif (F and G), and loop 3(H).

Fig. 2. Interface between the converter domain of myosin and myosin ELC.

3D map of actomyosin complex filtered to 7 Å resolution shows the full size ELC (cyan) and traces of the RLC (green letters). A portion of bovine cardiac motor domain (residues 1–130, 647–806) along with the bound ELC (PDB: 6FSA) (Robert-Paganin et al., 2018) matches our density map (red arrow) to confirm that myosin R723 and R780 (blue atoms) of converter domain interact with E135, D136, E139 and R142 (orange atoms) of the myosin ELC (insert). Actin shown in tan, tropomyosin in black, myosin motor domain in plum, while ELC is cyan.

Fig. 3. Positioning of myosin pathological variants at the cardiac actomyosin interface.

(A) Atomic model of the cardiac actomyosin complex shows structural elements of cardiac myosin involved in interactions with actin and tropomyosin: CM loop (blue ribbons), loop 2 (red ribbons), loop 4 (yellow ribbons), HLH motif (cyan ribbons), and loop 3 (brown ribbons). Actin atoms are tan, tropomyosin is black, while myosin motor domain is plum. (B-G) Myosin pathogenic variants are in magenta, while residues involved in interaction with those variants are in green. The proposed interactions are indicated with colored arrows. Pathogenic variants are shown for CM loop (B), loop 2 (C), loop 4 (D), HLH motif (E and F), and loop 3 (G).

Fig. 4. Actin pathological variants.

Positioning of the seven actin regions that harbor actin missense mutations linked to cardiomyopathies is shown with black boxes on our model and is linked to the inserts as follows: actomyosin interface with CM loop and loop 2 (A), D-loop in SD2 of actin (B), two major helixes (78–92 and 112–126) in SD1 of actin (C), upper part of SD4 of actin (D), lower portion of SD3 of actin (E), Tm binding interface (F), and hydrophobic plug (HP) (G). Actin molecules in the two strands are either tan or grey, tropomyosin is black, while myosin is plum. The two views of the model are related by 180° rotation around actin helical axis. Actin pathogenic variants are in magenta, while their interacting partners are in green. The proposed interactions are marked with colored arrows.