Holding the reins on myosin V.

M yosins comprise a diverse family of molecular motor enzymes that use the energy from cycles of ATP binding, hydrolysis, and product release to perform mechanical work along actin filaments. Although all characterized myosins share a conserved catalytic motor domain, referred to as ‘‘head,’’ variations in enzymatic and structural properties allow different myosins to generate diverse types of motility (1). Muscle myosin is not processive; it tugs intermittently on actin filaments and remains dissociated much of the time so multiple myosins are needed to sustain constant movement. In contrast, myosin V is processive, and an individual twoheaded motor molecule takes multiple 36-nm steps, each coupled to the consumption of a single ATP molecule, and walks unidirectionally along an actin filament for long distances without detaching (2, 3). Myosin V walks following an asymmetric hand-over-hand mechanism (4, 5), where the heads alternate leading and trailing positions along actin, analogous to the hands of a rope climber. The two heads of myosin V are hypothesized to exert pushing and pulling forces that modulate each other’s mechanochemical cycles and coordinate their stepping behavior (1, 6–8). In this issue of PNAS, Purcell et al. (9) examine the effects of external loads on the duration of the actin-attached states of myosin V by using optical tweezers to apply forward (push) or backward (pull) loads on single myosin V heads. They discover that a backward force, thought to mimic the force transmitted to the leading head from the attached trailing head of double-headed myosin V, slows the rate of actin detachment. A pushing force in the direction of motion, which may resemble the force transmitted to the trailing head by an attached leading head, has minimal effects on the lifetime of actin-attached states. The work demonstrates that an asymmetry exists between the myosin V heads when bound to actin, where a head in the leading position is more sensitive to load than one in a trailing position, and advances our understanding of how head–head coordination and intramolecular strain regulate structural and kinetic transitions of myosin V. The myosin ATPase cycle has been extensively characterized in solution in the absence of external loads (ref. 1 and Fig. 1A). ATP binding and hydrolysis cycle myosin through a series of conformational states that bind actin filaments strongly (attached) or weakly (detached) depending on the chemical state of the bound nucleotide. In the absence of nucleotide or with bound ADP, myosin V binds actin filaments strongly and dissociates from them very slowly, about once every 20–150 s (10, 11). ATP binding to myosin dissociates it rapidly from actin. Rapid ATP hydrolysis forms ADP and Pi, which remain bound noncovalently to detached myosin, and is associated with a conformational change in detached myosin that rotates the lightchain binding domain, or ‘‘lever arm,’’ to the prepower stroke state. Myosin with bound ADP and Pi is an unstable, high-energy intermediate, but it is kinetically very stable because it releases Pi very slowly. Actin binding accelerates Pi release from myosin–ADP–Pi, resulting in force production and a mechanical displacement as myosin relaxes and rotates the lever arm back to the postpower-stroke position. Subsequent release of bound ADP generates myosin (with no bound nucleotide) attached strongly to actin, ready to repeat the cycle with the binding of another ATP. When bound to actin, the trailing head of myosin V is in a postpower-stroke position, and the leading head is in a prepower-stroke position (12, 13). In the presence of saturating ATP and absence of load, ADP release limits ATP-induced dissociation from actin and dictates the overall myosin V ATPase cycling rate (10). Rate-limiting ADP release causes a single, cycling myosin V head to spend most of its ATPase cycle time strongly bound to actin and ADP (as AM.D shown in Fig. 1A). This high ‘‘duty ratio’’ is critical for processivity because it enables at least one of the two heads of a myosin V molecule undergoing a processive run to be strongly bound to actin at any time, ensuring that random thermal forces do not cause it to diffuse away from its track. Although a single head of myosin V spends a majority of its cycle time strongly bound to actin, processive motility requires both heads of myosin V, and single-headed myosin V takes only one step per encounter with actin filaments (14). Most models of myosin V processivity assume that the heads coordinate their