Myosin subfragment 1 structures reveal a partially bound nucleotide and a complex salt bridge that helps couple nucleotide and actin binding

Abstract

Structural studies of myosin have indicated some of the conformational changes that occur in this protein during the contractile cycle, and we have now observed a conformational change in a bound nucleotide as well. The 3.1-A x-ray structure of the scallop myosin head domain (subfragment 1) in the ADP-bound near-rigor state (lever arm =45 degrees to the helical actin axis) shows the diphosphate moiety positioned on the surface of the nucleotide-binding pocket, rather than deep within it as had been observed previously. This conformation strongly suggests a specific mode of entry and exit of the nucleotide from the nucleotide-binding pocket through the so-called "front door." In addition, using a variety of scallop structures, including a relatively high-resolution 2.75-A nucleotide-free near-rigor structure, we have identified a conserved complex salt bridge connecting the 50-kDa upper and N-terminal subdomains. This salt bridge is present only in crystal structures of muscle myosin isoforms that exhibit a strong reciprocal relationship (also known as coupling) between actin and nucleotide affinity.

Fig. 1.

Overview of the nucleotide-binding pocket. (A) ADP (blue) in the scallop myosin near-rigor structure adopts a partially bound conformation at the front door of the nucleotide-binding pocket. For comparison, the fully bound ADP from the S1–ADP·VO₄ complex in the scallop prepower-stroke structure (PDB ID code 1QVI) is shown in light gray. Also shown is an F_o – F_c simulated-annealing electron density map at a 1.0 σ contour level, calculated after omitting ADP and sulfate. Residues in switch I (red) and N321 (pink) from the 50-kDa upper subdomain that are involved in weak interactions (see text) with the ADP diphosphate moiety are shown in stick representation. The same residues from the prepower-stroke conformation structure are shown in light gray. The only major difference between the two structures in the nucleotide-binding pocket of the enzyme is that N321 (50-kDa upper subdomain) makes closer contact with the bound nucleotide in the prepower-stroke structure. The side chain of R127 and the hydrogen bond between its main-chain amide and the ADP ribose O₂′ are not shown for clarity. The P-loop and purine-binding loop are shown in cyan and brown, respectively. E184 (cyan sticks) and R128 (brown sticks) in the two loops form a salt bridge (dashed lines) that protects one face of the ADP base from solvent. (B) Corresponding view of the ScS1–SO₄ structure, with an F_o – F_c simulated-annealing electron density map at a 2.0 σ contour level, calculated after omitting the sulfate. The E184/R128 salt bridge is not formed in the absence of a nucleotide, supporting the role of R128 in nucleotide recruitment.

Fig. 2.

Rotations of the 50-kDa upper subdomain in the three conformational states of scallop myosin. The 50-kDa upper subdomain rotates as a rigid body with respect to the N-terminal subdomain, with the center of rotation near R236 of switch I. (A) Schematic representation of the different subdomains in the ScS1–ADP structure. Red arrow indicates rotation of the 50-kDa upper subdomain, which closes the 50-kDa cleft when myosin binds strongly to actin (5, 6). (B) A superposition of the N-terminal subdomains (light blue) shows the relative positions of the 50-kDa upper subdomains in the present ScS1–ADP near-rigor state (red), the internally uncoupled state (green), and the prepower-stroke state (purple) structures. A red arrow indicates the rotation of the 50-kDa upper subdomain in the plane of the paper. The 50-kDa lower subdomain, the converter, and the lever arm have been omitted for clarity. ADP from the ScS1–ADP structure is shown in a blue ball-and-stick representation. Residues involved in the complex salt bridge (E177, R236, and E675) between the 50-kDa upper and N-terminal subdomains are also shown in a ball-and-stick representation. (C) Close-up view of the complex salt bridge, which is well situated to stabilize the rotation of the 50-kDa upper subdomain. All but one of the salt links have favorable geometry to form hydrogen bonds, and these are shown as red dashed lines. The additional nonhydrogen-bonded salt link is shown as a black dashed line.