The kinetic mechanism of myosin V

Abstract

Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D., et al. (1999) Nature (London) 400, 590-593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin.

Scheme 1

Figure 1

Kinetics of myosin V-1IQ and myosin V-1IQ-ADP binding to actin filaments. (A Inset) Time course of fluorescence change after mixing 1.2 μM pyrene actin filaments with 0.12 μM myosin V-1IQ (curve b) or buffer alone (curve a). The smooth lines through the raw data are the best fits to single exponentials (k_obs = 107 s⁻¹ for curve b). (A) Dependence of k_obs on actin filament concentration. Error bars represent ± 1 SD (n = 3–6). (B Inset) Time course of fluorescence change after mixing 1.7 μM pyrene actin filaments with 0.15 μM myosin V-1IQ-ADP (curve b) or buffer alone (curve a). k_obs = 8.2 s⁻¹ for curve b. (B) Dependence of k_obs on actin filament concentration.

Figure 2

MgATP-induces population of the weakly bound states. Dependence of k_obs on MgATP concentration. Error bars represent ± 1 SD (n = 3–5 from four different preparations). (Inset) Time course of fluorescence enhancement after mixing 30 μM MgATP with 1.25 μM pyrene acto-myosin V-1IQ. The smooth line is the best fit to a single exponential with a k_obs of 29 s⁻¹.

Figure 3

Kinetics of MgATP binding to myosin V-1IQ by tryptophan fluorescence. Dependence of k_obs on MgATP concentration. Points are the average of 1–3 transients. (Inset) Time course of intrinsic tryptophan fluorescence change after mixing 0.5 μM myosin V-1IQ with 200 μM MgATP (curve a) or buffer alone (curve b). The smooth line is the best fit to a single exponential with a k_obs of 219 s⁻¹.

Figure 4

Kinetics of MgATP hydrolysis by quench-flow. Time course of ADP-P_i formation after mixing 3.2 μM myosin V-1IQ in the presence (●) or absence (○) of 7.5 μM actin filaments with 100 μM MgATP. The smooth lines are the best fits to a single exponentials with a slope. Points are the average of 2–3 P_i determinations.

Figure 5

Kinetics of mantADP binding to myosin V-1IQ and acto-myosin V-1IQ. Dependence of mantADP binding to myosin V-1IQ (○) or acto-myosin V-1IQ (●). The k_obs were determined from the fits of the time courses to single exponentials. (Inset) Time course of mantADP dissociation from acto-myosin V-1IQ (curve AM) and myosin V-1IQ (curve M). The rate constants for mantADP dissociation (k₋) determined from fits to single exponentials are 1.24 (± 0.01) s⁻¹ for myosin V-1IQ and 11.7 (± 0.2) s⁻¹ for acto-myosin V-1IQ. Fluorescence is normalized for clarity. Points are the average of 2–4 transients.

Figure 6

ADP inhibits population of the weakly bound states induced by MgATP. The rates of pyrene fluorescence increase after mixing 300 μM (●), 700 μM (○), or 1.4 mM (▴) MgATP with 0.5 μM acto-myosin V-1IQ equilibrated with the indicated [MgADP] (after mixing). Points are the average of 1–3 transients. The smooth lines through the data in the inset are simulations generated as described in the text.

Figure 7

Determination of the rate constant for transition from weakly bound to strongly bound states by sequential mixing. Measurements and simulations (smooth lines) were made as described in the text. Fluorescence under rigor conditions is set to zero (not shown for clarity). Final conditions at t = 0 sec were 2.3 μM myosin V-1IQ, 7.5 μM pyrene-actin, 100 μM MgATP, 2 mM MgADP (lower trace only), 25°C.

Figure 8

Kinetics of P_i release from 1IQ as a function of actin concentration. Myosin V 1IQ (4.0 μM) was mixed with 1 mM MgATP, aged for 14 ms to populate the weak-binding states then mixed with a solution of actin filaments and P_i-binding protein. Time courses fit two exponentials. The fast rate (●) corresponds to a phosphate burst during the first turnover of ATP with a stoichiometry of one P_i/myosin and the slow rate (○) to yields the steady-state ATPase rate at 1 μM ADP. Final concentrations at t = 0 were 1.0 μM 1IQ, 250 μM MgATP, 10 μM P_i-binding protein, and the indicated actin filament concentrations.