Structure of the Rigor Actin-Tropomyosin-Myosin Complex

Abstract

The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. Myosin converts the chemical energy stored in ATP into force and movement along actin filaments. Myosin binding to actin induces conformational changes that are coupled to the nucleotide-binding pocket and amplified by a specialized region of the motor domain for efficient force generation. Tropomyosin plays a key role in regulating the productive interaction between myosins and actin. Here, we report the 8 Å resolution structure of the actin-tropomyosin-myosin complex determined by cryo electron microscopy. The pseudo-atomic model of the complex obtained from fitting crystal structures into the map defines the large actin-myosin-tropomyosin interface and the molecular interactions between the proteins in detail and allows us to propose a structural model for tropomyosin dependent myosin binding to actin and actin-induced nucleotide release from myosin.

Force-producing ADP state of myosin bound to actin

“…When the first structure of myosin in a Rigorlike state was obtained (2), the structural basis for the long-known reciprocal nature of either strong actin or strong nucleotide binding became immediately evident (11). These insights have been confirmed with the highest-resolution actomyosin Rigor EM maps that have been published afterward (12,13). Strong binding to actin requires closure of the major cleft between the subdomains of the myosin motor to form a strong actin-binding interface, which in turn results in a relative movement of the subdomains of the myosin motor that contain the nucleotide-binding elements, Switch I and the P-loop.…”

“…S4). There has been some controversy about the role of this loop for binding to F-actin in Rigor (12,13,23). Here, the C-terminal basic residues of this loop seem to be in position to interact with D24, D25 of subdomain 1 of F-actin.…”

Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin

“…Because of the necessity of applying the helical symmetry of actomyosin to achieve maximum resolution, the molecular ends of Tm as well as the axial and rotational position of Tm with respect to actomyosin are not defined in the EM structure. The Tm molecule in the pseudoatomic model of the rigor actin-Tm-myosin S1 complex showed electrostatic interactions of Tm residues in the first half of P5 with both actin and myosin (10), and fewer interactions of Tm residues in the second-half of the period (Fig. 5A).…”

“…The radial distance between Tm and F-actin is 40 Å (10). Interactions of Tm with actin (green) and myosin (red) are shown in P3-P6: (A) EM structure of actin-Tm-myosin S1 (PDB ID 4A7F) (10) showing few interactions of the mutated Tm residues in P3-P4 with actin and myosin, and (B and C) crystal structure of Tm fragments including P3-P4 (PDB ID 2B9C) (B), and P5-P6 (PDB ID 2D3E) (C) (33,34) positioned in the EM structure of actin-Tm-myosin S1 (PDB ID 4A7F), showing more optimal electrostatic interactions of mutated Tm residues with actin and myosin (Movie S1). The Tm molecule in the EM structure is shown in gray and the crystal structures of Tm that were positioned in the EM structure are shown in blue (B and C).…”

“…The results provide a basis for establishing models for binding of Tm to the actin filament in the different regulatory states and the residues involved in switching it from one state to another. We have constructed a molecular model of Tm bound to F-actin in the open state by docking high-resolution Tm structures (33,34) to a proposed binding site in a 8 Å rigor actin-Tm-myosin S1 complex determined by cryo-EM (10). Because of the necessity of applying the helical symmetry of actomyosin to achieve maximum resolution, the molecular ends of Tm as well as the axial and rotational position of Tm with respect to actomyosin are not defined in the EM structure.…”