Structural mechanism of the ATP-induced dissociation of rigor myosin from actin

Abstract

Myosin is a true nanomachine, which produces mechanical force from ATP hydrolysis by cyclically interacting with actin filaments in a four-step cycle. The principle underlying each step is that structural changes in separate regions of the protein must be mechanically coupled. The step in which myosin dissociates from tightly bound actin (the rigor state) is triggered by the 30 Å distant binding of ATP. Large conformational differences between the crystal structures make it difficult to perceive the coupling mechanism. Energetically accessible transition pathways computed at atomic detail reveal a simple coupling mechanism for the reciprocal binding of ATP and actin.

Fig. 1.

Rigor dissociation. (A) The Lymn–Taylor cycle. In the rigor-dissociation step (I → II), ATP binding to the myosin head in the rigor state (I) is coupled to dissociation from the F-actin fibril (olive spheres), going to the postrigor state (II). Step II → III: Priming (back swing) of the lever arm (in yellow) is coupled to activation of the ATPase (–19). Step III → III^′: ATP-hydrolysis is coupled to an affinity-increase for actin (20). Step III^′ → I: Rebinding to actin is coupled to the force-generating swing of the lever arm (36, 37). (B) Rigor conformation of the myosin head (9) showing the two regions (circled in black) that contact F actin (in olive, not present in the calculations) (10). One region (in red) belongs to the u50 domain (in pink), which is organized around helix O (shown as cylinder). The other region (in black) belongs to the lower 50-kDa domain (part of the central body, in gray). The ATP binding site is between the switch-1 loop (in purple) and the P loop (in orange, Fig. 2B). The converter domain is shown in green, the central β-sheet in blue (same color code used throughout this article, except in Fig. 1C). (C) An intermediate taken one-third along the calculated transition pathway (in pink, blue, cyan, and purple) is very similar to the crystal structure of a semirigor conformer of myosin II (in yellow) determined by Reubold et al. (6). The switch-1 loop and the axis of helix O are also shown for the rigor (in gray) and the postrigor (in white) crystal structures of myosin V. All structures are oriented by a best fit of the central body (helix F, relay helix, and central β-sheet).

Fig. 2.

The coupling mechanism. Left panels show the rigor state I; right panels show the postrigor state II; thick arrows indicate motions during the transition (see Movies S1 and S2, and http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer for videos in other views). F actin (in olive) is shown for orientation; it was not present in the calculations. (A) The u50 domain (pink/red) and the attached switch-1 loop (purple) cohesively rotate by a 13° scissor motion relative to the central body (in gray/black), thereby twisting the central β-sheet (in blue) that connects the u50 domain to the central body. The scissor axis (determined by DynDom, ref. 38) is shown as a thick black rod. (B) This actin-binding cleft opens (5 → 16.4 Å), thus lowering the affinity for actin, and sandwiches ATP between the switch 1 and the P loops, thus stabilizing the postrigor state. (C and D) Same as A and B, in cartoon representation. The scissor axis (blue dotted line) passes through the central β-sheet (blue cylinder).

Fig. 3.

Measurements along the transition. (A–C) Correlated behavior of the u50 domain, helix O, the switch-1 loop, and actin-binding cleft. (A) Pathway with prebound ATP (Methods). Rotation angles of the u50 domain and of helix O relative to the central body: left plot scale. Displacement of the switch-1 loop: right plot scale. (B) Same as A, but for the pathway with ATP binding later in the transition. (C) Twisting angle of the central β-sheet: left scale. Actin-binding cleft opening (5 → 16.4 Å in Fig. 2 A and B) and strut length: right scale. (D–F) H bonds made by switch 1 in the ATP binding site. Dotted curves indicate transient H bonds; solid curves show the final H bonds of the postrigor state. (D) Distance from the Ser218 side-chain oxygen to the Mg²⁺ ion and to one γ-phosphate oxygen. (E) Distance from the backbone nitrogen of Ser218 to γ-phosphate oxygens. (F) Distance from the Ser217 side-chain oxygen to one γ-phosphate oxygen and to the side-chain oxygen of Ser165 (P loop).

Fig. 4.

Switch-1/ATP interactions. (A) Starting rigor structure with ATP (phosphorous colored gold) placed outside its binding pocket. The cyan arrow indicates the motion of ATP when it binds (Movie S4). (B) Starting rigor structure with ATP inside its pocket. The concerted rotation of helix-G axis (black arrow) and switch-1 loop (purple arrow) accompany the scissor motion of the u50 domain (Movie S3). (C) Halfway along the pathway, ATP found its final position with respect to the P loop. Relative to their initial position in A or B, switch 1 and helix G have pivoted 6°, switch 1 partially interacts with the Mg/triphosphate (as summarized in Fig. S2A). (D) The final (postrigor) state: Helix G is rotated by 12°. The switch-1 loop fully covers the triphosphate, Ser217/218 forming key interactions with the γ-phosphate/Mg²⁺ (Fig. S2B).