Functional role of loop 2 in myosin V.

Myosin V is molecular motor that is capable of moving processively along actin filaments. The kinetics of monomeric myosin V containing a single IQ domain (MV 1IQ) differ from nonprocessive myosin II in that actin affinity is higher, phosphate release is extremely rapid, and ADP release is rate- limiting. We generated two mutants of myosin V by altering loop 2, a surface loop in the actin-binding region thought to alter actin affinity and phosphate release in myosin II, to determine the role that this loop plays in the kinetic tuning of myosin V. The loop 2 mutants altered the apparent affinity for actin (KATPase) without altering the maximum ATPase rate (VMAX). Transient kinetic analysis determined that the rate of binding to actin, as well as the affinity for actin, was dependent on the net positive charge of loop 2, while other steps in the ATPase cycle were unchanged. The maximum rate of phosphate release was unchanged, but the affinity for actin in the M‚ADP‚Pi-state was dramatically altered by the mutations in loop 2. Thus, loop 2 is important for allowing myosin V to bind to actin with a relatively high affinity in the weak binding states but does not play a direct role in the product release steps. The ability to maintain a high affinity for actin in the weak binding states may prevent diffusion away from the actin filament and increase the degree of processive motion of myosin V. Myosins make up a large superfamily of motor proteins that are capable of using the chemical energy from ATP hydrolysis to power the directed movement on actin filaments and function in a wide variety of cellular processes from muscle contraction to organelle transport (1). During the actomyosin ATPase cycle myosin shifts between actin- detached (weak-binding) and actin-attached states (strong- binding), and force generation occurs through a conforma- tional change in myosin during the transition from the weak- to the strong-actin binding states (reviewed in ref 2). Interestingly, the overall structure of myosin proteins appears to be quite conserved, while their biochemical and kinetic properties are quite divergent. It has been proposed that sequence variability in the two surface loops of myosin, which are susceptible to proteolysis and divide myosin into three domains (25, 50, and 20 kDa, Figure 1), may play a role in kinetically tuning a particular myosin to perform specific cellular functions ( 3-6). In the current study, we examine the role of loop 2, a surface loop in the actin-binding region of myosin, in kinetically tuning myosin V, a non- muscle myosin that functions as an organelle transporter. Myosin V has several unique biochemical features that allow it to move processively along actin (7), take multiple steps along actin without diffusing away, and function as an organelle transporter (8). The kinetics of myosin V are different from nonprocessive muscle myosin II in that ADP release in myosin V is slow and rate-limiting, while phosphate release is fast (9-11). This allows myosin V to populate the strong-binding states for a greater fraction of its ATPase cycle and increase its duty ratio, fraction of ATPase cycle that myosin is strongly bound to actin. In a previous report, we determined that myosin V has a higher affinity for actin in the weak binding states ( 12) than nonprocessive class II myosins, which may also be an important aspect of its ability to move processively. Thus, the current study is focused on examining the structural aspects of myosin V that give rise to its high affinity for actin.