Effect of phosphorylation on the binding of smooth muscle heavy meromyosin X ADP to actin

Relaxation of both smooth and skeletal muscles appears to be caused primarily by inhibition of the step associated with Pi release in the actomyosin ATPase cycle, rather than by a block in the binding of the myosin X ATP and myosin X ADP X Pi complexes to actin. In skeletal muscle, troponin-tropomyosin not only causes marked inhibition of Pi release, but it also markedly inhibits the binding of myosin subfragment-1 X ADP to actin, raising the possibility that the two phenomena are coupled in some way. In the present study we determined whether phosphorylation of smooth muscle heavy meromyosin (HMM) also affects both the binding of HMM X ADP to actin and the Pi release step. This was done by having phosphorylated and unphosphorylated HMM X ADP compete for sites on F-actin. At mu = 30 mM, phosphorylation increased the affinity of the HMM molecule for actin about 12-fold and at mu = 170 mM, there was less than a 3-fold increase in the affinity of HMM. If phosphorylation affects the binding of each head of HMM to the same extent, then phosphorylation caused about a 4- and 2-fold increase in the affinity of each head of HMM for actin at mu = 30 and 170 mM, respectively. In contrast, at both ionic strengths, phosphorylation caused more than 100-fold actin activation of the ATPase activity of smooth muscle HMM. Therefore, the marked activation of Pi release in the acto X HMM ATPase cycle upon phosphorylation of HMM is not accompanied by a comparable increase in the affinity of HMM X ADP for actin. We have also found that phosphorylation increases by only 4-fold the rate of Pi release from HMM alone. These results suggest that in smooth muscle, phosphorylation accelerates the step associated with the release of Pi both in the forward and the reverse direction without correspondingly affecting the binding of myosin X ADP to actin.     

The tail domain of myosin Va modulates actin binding to one head

Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va.ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va.ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain.     

Millisecond-scale biochemical response to change in strain

Muscle fiber contraction involves the cyclical interaction of myosin cross-bridges with actin filaments, linked to hydrolysis of ATP that provides the required energy. We show here the relationship between cross-bridge states, force generation, and Pi release during ramp stretches of active mammalian skeletal muscle fibers at 20°C. The results show that force and Pi release respond quickly to the application of stretch: force rises rapidly, whereas the rate of Pi release decreases abruptly and remains low for the duration of the stretch. These measurements show that biochemical change on the millisecond timescale accompanies the mechanical and structural responses in active muscle fibers. A cross-bridge model is used to simulate the effect of stretch on the distribution of actomyosin cross-bridges, force, and Pi release, with explicit inclusion of ATP, ADP, and Pi in the biochemical states and length-dependence of transitions. In the simulation, stretch causes rapid detachment and reattachment of cross-bridges without release of Pi or ATP hydrolysis.     

Mechanism of regulation of Acanthamoeba myosin-IC by heavy-chain phosphorylation

The ATPase activity of myosin-Is from lower eukaryotes is activated by phosphorylation by the p21-activated kinase family at the TEDS site on an actin-binding surface-loop. This actin-binding loop is the site of a cardiac myosin-II mutation responsible for some forms of familial hypertrophic cardiomyopathy. To determine the mechanism of myosin-I regulation by heavy-chain phosphorylation (HCP) and to better understand the importance of this loop in the function of all myosin isoforms, we performed a kinetic investigation of the regulatory mechanism of the Acanthamoeba myosin-IC motor domain (MIC(IQ)). Phosphorylated and dephosphorylated MIC(IQ) show actin-activated ATPase activity; however, HCP increases the ATPase activity >20-fold. HCP does not greatly affect the rate of phosphate release from MIC in the absence of actin, as determined by single turnover experiments. Additionally, HCP does not significantly affect the affinity of myosin for actin in the absence or presence of ATP, the rate of ATP-induced dissociation of actoMIC(IQ), the affinity of ADP, or the rate of ADP release. Sequential-mix single-turnover experiments show that HCP regulates the rate of phosphate release from actin-bound MIC(IQ). We propose that the TEDS-containing actin-binding loop plays a direct role in regulating phosphate release and the force-generating (A-to-R) transition of myosin-IC.     

The effect of F-actin on the relay helix position of myosin II, as revealed by tryptophan fluorescence, and its implications for mechanochemical coupling

The fluorescence properties of Dictyostelium discoideum (Dd) myosin II constructs containing a single tryptophan residue have revealed detailed information regarding nucleotide binding and hydrolysis steps. Here we extend these studies to investigate the influence of actin on nucleotide-induced fluorescence transients. The fluorescence from native actin tryptophan residues is not significantly perturbed on binding to myosin, although an apparent signal is detected as a consequence of a light scatter artifact. Actin has a minor effect on the response of W129, located at the entrance to the nucleotide-binding pocket, and reduces the forward rate constants for the isomerization(s) associated with binding of ATP, ATPgammaS, and ADP by 3-fold or less. The isomerization detected by W129 clearly precedes the dissociation of actin in the case of ADP and ATPgammaS binding. The fluorescence from the conserved W501 residue, located at the distal end of the relay helix, is very sensitive to the switch 2 and/or lever arm disposition. Consequently, the observed fluorescence emission intensity can be used to estimate the equilibrium constant between the pre- and post-power stroke conformations. Actin modulates this equilibrium by no more than 2-fold in the presence of nucleoside triphosphate. These data have implications for the mechanism of product release and suggest that actin activates another process in the mechanism, such as switch 1 movement and Pi release, rather than influencing the switch 2 equilibrium and lever arm position directly.     

A kinetic model that explains the effect of inorganic phosphate on the mechanics and energetics of isometric contraction of fast skeletal muscle

A conventional five-step chemo-mechanical cycle of the myosin–actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding of a new ATP molecule, is able to fit the [Pi] dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in [Pi] much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with [Pi]. The large dissociation constant of the product release in the branched pathway allows the isometric myosin–actin reaction to fit the equilibrium constant of the ATPase.     

Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction.

The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 microM actin decreased ADP affinity from 0.6 to 34 microM for M765NL and from 1.6 to 39 microM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for M765 (0.06 s-1), and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP and Pi release, and they confirm that loop 2 is involved in high affinity actin binding.     

A structural model for actin-induced nucleotide release in myosin

Myosins are molecular motor proteins that harness the chemical energy stored in ATP to produce directed force along actin filaments. Complex communication pathways link the catalytic nucleotide-binding region, the structures responsible for force amplification and the actin-binding domain of myosin. We have crystallized the nucleotide-free motor domain of myosin II in a new conformation in which switch I and switch II, conserved loop structures involved in nucleotide binding, have moved away from the nucleotide-binding pocket. These movements are linked to rearrangements of the actin-binding region, which illuminate a previously unobserved communication pathway between the nucleotide-binding pocket and the actin-binding region, explain the reciprocal relationship between actin and nucleotide affinity and suggest a new mechanism for product release in myosin family motors.     

Magnesium regulates ADP dissociation from myosin V

Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.     

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.     

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 (K(ATPase)) without altering the maximum ATPase rate (V(MAX)). 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.     

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.     

Magnesium, ADP, and actin binding linkage of myosin V: evidence for multiple myosin V-ADP and actomyosin V-ADP states

The [Mg(2+)] dependence of ADP binding to myosin V and actomyosin V was measured from the fluorescence of mantADP. Time courses of MgmantADP dissociation from myosin V and actomyosin V are biphasic with fast observed rate constants that depend on the [Mg(2+)] and slow observed rate constants that are [Mg(2+)]-independent. Two myosin V-MgADP states that are in reversible equilibrium, one that exchanges nucleotide and cation slowly (strong binding) and one that exchanges nucleotide and cation rapidly (weak binding), account for the data. The two myosin V-MgADP states are of comparable energies, as indicated by the relatively equimolar partitioning at saturating magnesium. Actin binding lowers the affinity for bound Mg(2+) 2-fold but shifts the isomerization equilibrium approximately 6-fold to the weak ADP binding state, lowering the affinity and accelerating the overall rate of MgADP release. Actin does not weaken the affinity or accelerate the release of cation-free ADP, indicating that actin and ADP binding linkage is magnesium-dependent. Myosin V and myosin V-ADP binding to actin was assayed from the quenching of pyrene actin fluorescence. Time courses of myosin V-ADP binding and release are biphasic, consistent with the existence of two (weak and strong) quenched pyrene actomyosin V-ADP conformations. We favor a sequential mechanism for actomyosin V dissociation with a transition from strong to weak actin-binding conformations preceding dissociation. The data provide evidence for multiple myosin-ADP and actomyosin-ADP states and establish a kinetic and thermodynamic framework for defining the magnesium-dependent coupling between the actin and nucleotide binding sites of myosin.     

Three myosin V structures delineate essential features of chemo-mechanical transduction

The molecular motor, myosin, undergoes conformational changes in order to convert chemical energy into force production. Based on kinetic and structural considerations, we assert that three crystal forms of the myosin V motor delineate the conformational changes that myosin motors undergo upon detachment from actin. First, a motor domain structure demonstrates that nucleotide-free myosin V adopts a specific state (rigor-like) that is not influenced by crystal packing. A second structure reveals an actomyosin state that favors rapid release of ADP, and differs from the rigor-like state by a P-loop rearrangement. Comparison of these structures with a third structure, a 2.0 angstroms resolution structure of the motor bound to an ATP analog, illuminates the structural features that provide communication between the actin interface and nucleotide-binding site. Paramount among these is a region we name the transducer, which is composed of the seven-stranded beta-sheet and associated loops and linkers. Reminiscent of the beta-sheet distortion of the F1-ATPase, sequential distortion of this transducer region likely controls sequential release of products from the nucleotide pocket during force generation.     

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å² to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V_max) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K_app) than that of the wild-type actin, with the V_max being almost unchanged. The K_app and V_max of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K_app was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/K_app reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.     

Probing actomyosin interactions with 2,4-dinitrophenol

Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10(-2) M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]-->infinity. In skinned fibers, isometric force is reduced to the same extent (K0.5 approximately equal to 6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 degrees C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10(-2) M) increases the apparent affinity for Pi.     

Mechanokinetics of rapid tension recovery in muscle: the Myosin working stroke is followed by a slower release of phosphate

Crystallographic and biochemical evidence suggests that the myosin working stroke that generates force in muscle is accompanied by the release of inorganic phosphate (Pi), but the order and relative speed of these transitions is not firmly established. To address this problem, the theory of A. F. Huxley and R. M. Simmons for the length-step response is averaged over elastic strains imposed by filament structure and extended to include a Pi-release transition. Models of this kind are applied to existing tension-recovery data from length steps at different phosphate concentrations, and from phosphate jumps upon release of caged phosphate. This body of data is simulated by the model in which the force-generating event is followed by Pi release. A version in which the Pi-release transition is slow provides a better fit than a version with rapid Pi release and a slow transition preceding force generation. If Pi is released before force generation, the predicted rate of slow recovery increases with the size of the step, which is not observed. Some implications for theories of muscle contraction are discussed.     

Force generation, work, and coupling in molecular motors.

A mechanism is proposed for molecular motors in which force is generated by a protein conformational change driven by binding energy (in muscle, that of myosin with actin as well as with ATP, ADP, or Pi). Work, the product of the force generated by one myosin or kinesin molecule (F) and the distance over which it acts (d), is a function of a ratio of dissociation constants before and after the contractile step: F.d < RT ln(KAe/KAc). From published data the ratio is > 2 x 10(4), which can be explained by conversion of a surface complex to an enclosed, or partly enclosed, complex. Although the complex performing the work stroke is in unstrained conformation, the complex after the work stroke is much more stable, owing to binding forces; the latter, however, is destabilized by the load, which thereby opposes the contractile conformational change, countering the force-generating reaction. The connection between the free energy release and work is implicit in the mechanism, inasmuch as coupling, like force generation, depends on conformational changes driven by binding energy (internal rather than external work being involved in coupling). The principles apply whether ATP or an ion gradient drives the system. At high load, in muscle, the mechanism allows for a summation of the forces generated by several myosin molecules.     

Smooth muscle myosin: regulation and properties

The relationship of the biochemical states to the mechanical events in contraction of smooth muscle cross-bridges is reviewed. These studies use direct measurements of the kinetics of Pi and ADP release. The rate of release of Pi from thiophosphorylated cycling cross-bridges held isometric was biphasic with turnovers of 1.8 s-1 and 0.3 s-1, reflecting properties and forces directly acting on cross-bridges through mechanisms such as positive strain and inhibition by high-affinity MgADP binding. Fluorescent transients reporting release of an ADP analogue 3'-deac-edaADP were significantly faster in phasic than in tonic smooth muscles. Thiophosphorylation of myosin regulatory light chains (RLCs) increased and positive strain decreased the release rate around twofold. The rates of ADP release from rigor cross-bridges and the steady-state Pi release from cycling isometric cross-bridges are similar, indicating that the ADP-release step or an isomerization preceding it may limit the ATPase rate. Thus ADP release in phasic and tonic smooth muscles is a regulated step with strain- and dephosphorylation-dependence. High affinity of cross-bridges for ADP and slow ADP release prolong the fraction of the duty cycle occupied by strongly bound AM.ADP state(s) and contribute to the high economy of force that is characteristic of smooth muscle. RLC thiophosphorylation led to structural changes in smooth muscle cross-bridges consistent with our findings that thiophosphorylation and strain modulate product release.     

Mechanism of nucleotide binding to actomyosin VI: evidence for allosteric head-head communication

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K(1)' = 5.6 mm) followed by isomerization (k(+2)' = 176 s-1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is approximately five times greater than for ATP binding because of a higher affinity in the collision complex (1/K(5b)' = 2.2 mm) and faster isomerization rate constant (k(+5a)' = 366 s(-1)). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.