SUMMARY Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential to use the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin, and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement, and may represent a strategy common to many molecular machines.

Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5°rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.T he actin-based molecular motor, myosin, generates force and movement via a series of structural transitions when bound to filamentous actin (F-actin). Among these structural states, the most important contribution to force production comes from the myosin ADP states that bind strongly to actin. Release of ADP from the motor is rapidly followed by MgATP binding, which then leads to myosin dissociation from actin. Once the dissociation occurs, myosin can undergo a structural change that allows it to prime its mechanical element (known as the lever arm; Fig. 1) and hydrolyze ATP. Myosin rebinding to actin triggers release of phosphate and reentry into the force-generating states on actin. Thus, force is produced by MgADP-bound states of myosin bound to actin.In the absence of strain, a strong-binding MgADP state bound to actin can exist for varying durations, depending on the type of myosin. Myosins that are designed to spend the majority of their catalytic cycle bound to actin in force-generating states in the absence of load, such as myosin V a , primarily occupy a state that is characterized by having both a high affinity for MgADP and a high affinity for actin. The myosin motor cycle is summarized in Fig. 1. By not rapidly releasing MgADP, the myosin motors can dwell in a force-generating state on actin that cannot be detached from actin by ATP binding. This is the primary force-generating state for all myosins, and its lifetime on actin is prolonged under load (for a review, see ref. 1).Although the nucleotide-free myosin V X-ray structure (2) (Rigor-like state) provides an atomic level model for the actomyosin state that is formed after ADP is released, and to which ATP can rapidly bind (2), we only have low-resolution EM reconstructions of myosin bound to actin in a MgADP state. Visualization of this state at the EM level was first performed for smooth muscle myosin II (3) and provided the first structural evidence that the myosin lever arm swings as the motor progresses through its actinbound, force-generating states. It was postulated at the time that th...