Direct chemical evidence that serine 180 in the glycine-rich loop of myosin binds to ATP

The photochemical reaction of MgADP-vanadate with the active site of myosin has been used to place a serine at the binding site for the gamma-phosphate of ATP. Irradiation of the MgADP-vanadate myosin subfragment 1 transition state-like complex with UV light specifically photooxidizes the hydroxyl group of a serine residue to an aldehyde (Cremo, C. R., Grammer, J. C., and Yount, R. G. (1988) Biochemistry 27, 8415-8420). Reduction of photooxidized myosin with Na-B3H4 gave only 3H-labeled serine. Here, subsequent extensive proteolytic digestion of 3H-labeled myosin subfragment 1 with trypsin and thermolysin yielded two 3H-labeled peptides, both of which contained the sequence Gly-Glu-Ser-Gly-Ala-Gly-Lys-Thr, in which all the 3H was associated with the serine. This sequence is conserved in all myosin heavy chains sequenced to date and corresponds to residues 178-185 in the rabbit myosin heavy chain (Tong, S. W., and Elzinga, M. (1983) J. Biol. Chem. 21, 13100-13110). These results place Ser-180 at the gamma-phosphate-binding site for ATP and indicate that the glycine-rich loop around the serine provides essential elements of the phosphate-binding site for ATP in all myosin molecules. Such a role was previously suggested based on the common sequence Gly-X-X-X-X-Gly-Lys-Thr/Ser, found in myosin and many other nucleotide-binding enzymes (Walker, J. E., Saraste, M., Runswick, M. H., and Gay, N. J. (1982) EMBO J. 1, 945-951).     

A classical and ab initio study of the interaction of the myosin triphosphate binding domain with ATP.

We used classical molecular mechanics (MM) simulations and quantum mechanical (QM) structural relaxations to examine the active site of myosin when bound to ATP. Two conformations of myosin have been determined by x-ray crystallography. In one, there is no direct interaction between switch 2 and the nucleotide (open state). In the other (closed state), the universally conserved switch 2 glycine forms a hydrogen bond with a gamma-phosphate oxygen. MM simulations indicate that the two states are thermodynamically stable and allow us to investigate the extent to which the P-loop, switch 1, and switch 2 are involved in hydrolysis. We find that the open structure has a higher affinity for ATP than the closed structure, and that ATP is distorted toward a transition state by interactions with the protein. We also examine how the structure of the binding site changes with either MgATP or CaATP as the nucleotide in myosin in the open conformer. Our analyses suggest that higher CaATPase rates occur because the leaving phosphate (P(i)) group is more weakly bound and dissociation occurs faster. Finally, we validate the use of a particular formulation of a QM methodology (Car-Parrinello) to further refine the structures of the active site.     

Analysis of the conformational change of myosin during ATP hydrolysis using fluorescence resonance energy transfer

In order to elucidate the molecular basis of energy transduction by myosin as a molecular motor, a fluorescent ribose-modified ATP analog 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ATP (NBD-ATP), was utilized to study the conformational change of the myosin motor domain during ATP hydrolysis using the fluorescence resonance energy transfer (FRET) method. The FRET efficiency from the fluorescent probe, BD- or AD-labeled at the reactive cysteine residues, SH1 (Cys 707) or SH2 (Cys697), respectively, to the NBD fluorophore in the ATP binding site was measured for several transient intermediates in the ATPase cycle. The FRET efficiency was greater than that using NBD-ADP. The FRETs for the myosin.ADP.AlF4- and myosin.ADP.BeFn ternary complexes, which mimic the M*.ADP.P(i) state and M.ATP state in the ATPase cycle, respectively, were similar to that of NBD-ATP. This suggests that both the SH1 and SH2 regions change their localized conformations to move closer to the ATPase site in the M*.ATP state and M**.ADP.P(i) state than in the M*.ADP state. Furthermore, we measured energy transfer from BD in the essential light chain to NBD in the active site. Assuming the efficiency at different states, myosin adopts a conformation such that the light chain moves closer to the active site by approximately 9 A during the hydrolysis of ATP.     

Photochemical mapping of the active site of myosin.

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the gamma-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.     

Switch II mutants reveal coupling between the nucleotide- and actin-binding regions in myosin V

Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the γ phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide- and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin.ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.     

X-ray structures of the apo and MgATP-bound states of Dictyostelium discoideum myosin motor domain

Myosin is the most comprehensively studied molecular motor that converts energy from the hydrolysis of MgATP into directed movement. Its motile cycle consists of a sequential series of interactions between myosin, actin, MgATP, and the products of hydrolysis, where the affinity of myosin for actin is modulated by the nature of the nucleotide bound in the active site. The first step in the contractile cycle occurs when ATP binds to actomyosin and releases myosin from the complex. We report here the structure of the motor domain of Dictyostelium discoideum myosin II both in its nucleotide-free state and complexed with MgATP. The structure with MgATP was obtained by soaking the crystals in substrate. These structures reveal that both the apo form and the MgATP complex are very similar to those previously seen with MgATPgammaS and MgAMP-PNP. Moreover, these structures are similar to that of chicken skeletal myosin subfragment-1. The crystallized protein is enzymatically active in solution, indicating that the conformation of myosin observed in chicken skeletal myosin subfragment-1 is unable to hydrolyze ATP and most likely represents the pre-hydrolysis structure for the myosin head that occurs after release from actin.     

Localization of the ATP-binding site in the 23-kDa and 20-kDa regions of the heavy chain of the skeletal muscle myosin head

Three kinds of ATP analogues were synthesized. These ATP analogues can be classified into two conformations, i.e. syn and anti forms with respect to the N-glycosidic bond between adenine and ribose groups of ATP. 3'-O-(N-Methylanthraniloyl)-2-azidoadenosine 5'-triphosphate (MantN2(3)ATP) is recognized as the anti form, as ATP, and the other two, 3'-O-(N-methylanthraniloyl)-8-azidoadenosine 5'-triphosphate (MantN8(3)ATP) and 1,N6-etheno-8-azidoadenosine 5'-triphosphate (epsilon N8(3)ATP) are both syn forms. Mant and etheno groups are both fluorescent which allows detection of their binding to proteins. The photochemical binding of azido groups in ATP analogues to the myosin active site, examined in the presence and absence of ATP, showed that all the analogues bound to the site of myosin ATPase. These analogues also acted as substrates of the ATPase and were hydrolyzed in the active site, as judged by competitive inhibition of the ATPase and by their ATPase activities. Of these analogues, MantN2(3)ATP is very similar to ATP in divalent-cation dependence of its hydrolysis rate and in its ability to trap ADP in the active site with vanadate, while the other two are different from ATP in these respects. The photochemical binding sites of ATP analogues were localized by gel electrophoresis of trypsinized myosin ATPase with photocross-linked ATP analogues and/or by isolating the modified peptides. MantN2(3)ATP was found in the 23-kDa fragment which has a structure common to ATP-binding proteins, i.e. Gly-Xaa-Xaa-Gly-Xaa-Gly-Lys-Thr. Mant N8(3)ATP was found in a region of the 20-kDa fragment where actin is reported to attach.     

The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading

Replication factor C (RFC) is an AAA+ heteropentamer that couples the energy of ATP binding and hydrolysis to the loading of the DNA polymerase processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. RFC consists of five subunits in a spiral arrangement (RFC-A, -B, -C, -D, and -E, corresponding to subunits RFC1, RFC4, RFC3, RFC2, and RFC5, respectively). The RFC subunits are AAA+ family proteins and the complex contains four ATP sites (sites A, B, C, and D) located at subunit interfaces. In each ATP site, an arginine residue from one subunit is located near the gamma-phosphate of ATP bound in the adjacent subunit. These arginines act as "arginine fingers" that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. In this study, the arginine fingers in RFC were mutated to examine the steps in the PCNA loading mechanism that occur after RFC binds ATP. This report finds that the ATP sites of RFC function in distinct steps during loading of PCNA onto DNA. ATP binding to RFC powers recruitment and opening of PCNA and activates a gamma-phosphate sensor in ATP site C that promotes DNA association. ATP hydrolysis in site D is uniquely stimulated by PCNA, and we propose that this event is coupled to PCNA closure around DNA, which starts an ordered hydrolysis around the ring. PCNA closure severs contact to RFC subunits D and E (RFC2 and RFC5), and the gamma-phosphate sensor of ATP site C is switched off, resulting in low affinity of RFC for DNA and ejection of RFC from the site of PCNA loading.     

Inhibition of myosin ATPase by metal fluoride complexes

Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.     

The mechanism of the skeletal muscle myosin ATPase. I. Identity of the myosin active sites.

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-1 was determined by measuring the fluorescence enhancement caused by the binding of MgATP. The amount of irreversible ATP binding and the magnitude of the initial ATP hydrolysis (initial Pi burst) was determined by measuring [gamma-32P]ATP hydrolysis with and without a cold ATP chase in a three-syringe quenched flow apparatus. The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100% of this ATP binding is irreversible, 3) 70 to 90% of the irreversibly bound ATP is hydrolyzed in the initial Pi burst, 4) the first order rate constant for the rate-limiting step in ATP hydrolysis by heavy meromyosin is equal to the steady state heavy meromyosin ATPase rate only if the latter is calculated on the basis of two active sites per heavy meromyosin molecule. It is concluded that the two active sites of myosin are identical with respect to ATP binding and hydrolysis.     

Investigating the role of the invariant carboxylate residues E552 and E1197 in the catalytic activity of Abcb1a (mouse Mdr3)

The invariant carboxylate residue which follows the Walker B motif (hyd(4)DE/D) in the nucleotide-binding domains (NBDs) of ATP-binding cassette transporters is thought to be involved in the hydrolysis of the gamma-phosphate of MgATP, either by activating the attacking water molecule or by promoting substrate-assisted catalysis. In Abcb1a, this invariant carboxylate residue corresponds to E552 in NBD1 and E1197 in NBD2. To further characterize the role of these residues in catalysis, we created in Abcb1a the single-site mutants E552D, N and A in NBD1, and E1197D, N and A in NBD2, as well as the double-mutant E552Q/E1197Q. In addition, we created mutants in which the Walker A K --> R mutation known to abolish ATPase activity was introduced in the non-mutant NBD of E552Q and E1197Q. ATPase activity, binding affinity and trapping properties were tested for each Abcb1a variant. The results suggest that the length of the invariant carboxylate residue is important for the catalytic activity, whereas the charge of the side chain is critical for full turnover to occur. Moreover, in the double-mutants where the K --> R mutation is introduced in the 'wild-type' NBD of the E --> Q mutants, single-site turnover is observed, especially when NBD2 can undergo gamma-P(i) cleavage. The results further support the idea that the NBDs are not symmetric and suggest that the invariant carboxylates are involved both in NBD-NBD communication and transition-state formation through orientation of the linchpin residue.     

Mutational analysis of the functional motifs in the ATPase domain of Caenorhabditis elegans fidgetin homologue FIGL-1: firm evidence for an intersubunit catalysis mechanism of ATP hydrolysis by AAA ATPases

The AAA family proteins usually form a hexameric ring structure. The ATP-binding pocket, which is located at the interface of subunits in the hexamer, consists of three functionally important motifs, the Walker A and B motifs, and the second region of homology (SRH). It is well known that Walker A and B motifs mediate ATP binding and hydrolysis, respectively. Highly conserved arginine residues in the SRH have been proposed to function as arginine fingers, which interact with the gamma-phosphate of bound ATP. To elucidate the mechanism of ATP hydrolysis, we prepared several mutants of the Caenorhabditis elegans fidgetin homologue FIGL-1 carrying a mutation in each of the above-mentioned three motifs. None of the constructed mutants showed ATPase activity. All the mutants except for K362A were able to bind ATP. A decrease in the ATPase activity by mixing wild-type and each mutant subunits was caused by the formation of hetero-hexamers. Mixtures of E416A and R471A, or N461A and R471A led to the formation of hetero-hexamers with partially restored ATPase activities, providing direct, firm evidence for the intersubunit catalysis model. In addition, based on the results obtained with mixtures of K362A with wild-type or R471A subunits, we propose that a conformational change upon ATP binding is required for proper orientation of the arginine fingers, which is essential for efficient hydrolysis of ATP bound to the neighboring subunit.     

The Hypertrophic Cardiomyopathy Myosin Mutation R453C Alters ATP Binding and Hydrolysis of Human Cardiac β-Myosin

The human hypertrophic cardiomyopathy mutation R453C results in one of the more severe forms of the myopathy. Arg-453 is found in a conserved surface loop of the upper 50-kDa domain of the myosin motor domain and lies between the nucleotide binding pocket and the actin binding site. It connects to the cardiomyopathy loop via a long α-helix, helix O, and to Switch-2 via the fifth strand of the central β-sheet. The mutation is, therefore, in a position to perturb a wide range of myosin molecular activities. We report here the first detailed biochemical kinetic analysis of the motor domain of the human β-cardiac myosin carrying the R453C mutation. A recent report of the same mutation (Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., and Spudich, J. A. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 12607–12612) found reduced ATPase and in vitro motility but increased force production using an optical trap. Surprisingly, our results show that the mutation alters few biochemical kinetic parameters significantly. The exceptions are the rate constants for ATP binding to the motor domain (reduced by 35%) and the ATP hydrolysis step/recovery stroke (slowed 3-fold), which could be the rate-limiting step for the ATPase cycle. Effects of the mutation on the recovery stroke are consistent with a perturbation of Switch-2 closure, which is required for the recovery stroke and the subsequent ATP hydrolysis.     

Structural rearrangements in the active site of smooth-muscle myosin

Structural rearrangements of the myosin upper-50 kD subdomain are thought to play a key role in coordinating actin binding with nucleotide hydrolysis during the myosin ATPase cycle. Such rearrangements could open and close the active site in opposition to the actin-binding cleft, helping explain the opposing affinities of myosin for actin and nucleotide. To directly examine conformational changes across the active site during the ATPase cycle we have genetically engineered a mutant of chicken smooth-muscle myosin, F344W motor domain essential light chain, which contains a single tryptophan (344W) located on a short loop between two alpha helixes that traverse the upper-50 kD subdomain in front of the active site. Fluorescence resonance energy transfer was examined between the 344W donor probe and 2'(3')-O-(N-methylanthraniloyl) (mant)-nucleotide acceptor probes in the active site of this construct. The observed fluorescence resonance energy transfer efficiencies were 6.4% in the presence of mant ADP and 23.8% in the presence of mant ATP, corresponding to distances of 33.4 A and 24.9 A, respectively. Our results are consistent with structural rearrangements in which there is an 8.5-A closure between the 344W residue and the mant moiety during the transition from the strongly (ADP) to weakly (ATP) actin-bound states of the myosin ATPase cycle.     

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.     

Cryoenzymic studies on myosin: transient kinetic evidence for two types of head with different ATP binding properties

The two step tight binding of ATP to myosin, heavy meromyosin and myosin subfragment 1 was investigated, under cryoenzymic conditions by the unlabeled ATP chase method: M + ATP in equilibrium K1 M.ATP k2 in equilibrium k-2 M*.ATP where M is myosin. k-2 is close to zero. In multi-turnover experiments, one obtains the constants for the binding process together with the concentration of ATPase sites. Here the kinetics of the formation of M*.ATP are first order. Inversion of the reagent concentrations (i.e., single-turnover experiments) should give identical kinetics but such experiments often give biphasic curves. This biphasicity depends upon the myosin preparation used and it is directly related to the active site titration. The simplest explanation for these results is one involving 2 sites for ATP: one site hydrolyzes ATP by the Bagshaw-Trentham scheme (tight binding preceding hydrolysis) but the second site binds ATP loosely without significant hydrolysis. This heterogeneity in ATP binding may explain certain difficulties, such as questions concerning the non-identity of the myosin heads and the number of steps involved in nucleotide binding. Attempts were made to determine the cause of the head heterogeneity but these were unsuccessful. We cannot exclude the possibility that the heterogeneity is relevant to muscle contraction.     

On the myosin catalysis of ATP hydrolysis

Myosin is an ATP-hydrolyzing motor that is critical in muscle contraction. It is well established that in the hydrolysis that it catalyzes a water molecule attacks the gamma-phosphate of an ATP bound to its active site, but the details of these events have remained obscure. This is mainly because crystallographic search has not located an obvious catalytic base near the vulnerable phosphate. Here we suggest a means whereby this dilemma is probably overcome. It has been shown [Fisher, A. J., et al. (1995) Biochemistry 34, 8960-8972; Smith, C. A., and Rayment, I. (1996) Biochemistry 35, 5404-5417] that in an early event, Arg-247 and Glu-470 come together into a "salt-bridge". We suggest that in doing so they also position and orient two contiguous water molecules; one of these becomes the lytic water, perfectly poised to attack the bound gamma-phosphorus. Its hydroxyl moiety attacks the phosphorus, and the resulting proton transfers to the second water, converting it into a hydronium ion (as is experimentally observed). It is shown in this article how these central events of the catalysis are consistent with the behavior of several residues of the neighboring region.     

Synthesis of a novel fluorescent non-nucleotide ATP analogue and its interaction with myosin ATPase

A novel non-nucleotide fluorescent ATP analogue, N-methylanthraniloylamideethyl triphosphate (MANTTP), was designed and synthesized for kinetic studies with ATPases. The interaction of MANTTP with myosin ATPase was characterized. MANTTP was used as a substrate of myosin ATPase, and acceleration of actin-dependent hydrolysis was observed. The fluorescence property of MANTTP was not greatly affected by its binding to the ATPase site of myosin. In contrast, during MANTTP hydrolysis, significant fluorescence resonance energy transfer (FRET) was observed between MANTTP and intrinsic tryptophan residues in the myosin motor domain. Binding of MANTTP and formation of a ternary complex with a myosin-N-methylanthraniloylamideethyl diphosphate (MANTDP)-Pi analogue, which may mimic ATPase transient states, were monitored by FRET. The kinetic parameters of MANTTP binding to myosin and MANTDP release from the ATPase site were determined using a stopped-flow apparatus and compared with those of other ATP analogues. This novel fluorescent ATP analogue was shown to be applicable for kinetic analysis of ATPases.     

Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg²⁺) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg²⁺- or Mn²⁺-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn²⁺, providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn²⁺ rescues this effect to near wild-type activity. 2′(3′)-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region.