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Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, Vermont
Correspondence: Address reprint requests to Christopher L. Berger, Dept. of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, VT 05405. Tel.: 802-656-0832; Fax: 802-656-0747; E-mail: cberger{at}uvm.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). From the strongly actin-bound AM rigor complex, myosin binds tightly to ATP causing a decrease in its affinity for actin by >4 orders of magnitude, triggering actin release. After ATP hydrolysis, Pi release is thought to result in a conformational change in which myosin increases its affinity for actin from micromolar to nanomolar and initiates its power stroke. Myosin remains strongly bound to actin after ADP release, until a new molecule of ATP binds at the active site. To avoid confusion, we will refer to myosin as being strongly bound to actin but tightly bound to ATP throughout the text. Myosin has a high affinity for actin in the apo and ADP states (even in the absence of actin) and thus we will refer to these as strong-binding states throughout the text.
A wealth of myosin II crystal structures, solved in the absence of nucleotide and in the presence of nucleotides and nucleotide analogs (2–7), have allowed assignment of force production, actin binding, and nucleotide hydrolysis to specific structural domains (Fig. 1). The myosin force-generation function is thought to be provided by a lever-arm domain (residues 781–820), which has been observed in numerous positions in crystal structures of myosin. It has been proposed that the various positions all correlate to one of two conformational classes, either a pre-power stroke state in which the lever arm is tilted up relative to the core domain or a post-power stroke "near-rigor" state in which the lever arm is pointed down (8).
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) and undergoes some rearrangements in the recent myosin II apo structure of Reubold et al. (8). As myosin V binding to actin is diffusion-limited, it has been suggested that the myosin V actin-binding cleft is already closed in the apo state in the absence of actin, making myosin V an instructive special case. Fitting skeletal and smooth-muscle myosin crystal structures into cryoelectron microscopy (cryo-EM) density maps of S1-decorated actin filaments predicts a greater closing of the actin-binding cleft in the posthydrolysis state than is seen in any of the crystal structures (10,11). In fact, rotation of the upper- and lower-50-kD subdomains is seen in the recent myosin apo structures. For chicken smooth-muscle myosin II, a solution study using an intrinsic tryptophan probe located in the 50-kD cleft showed that actin-binding and closure of the actin-binding cleft occur at nearly identical rates (12). Fluorescence data from this study suggested cleft closure in strongly actin-bound states even in the absence of actin. This data supports a model of the transition from the weakly to the strongly actin-bound state in which the sum of many small attractions between myosin and actin produces movement of the upper-50-kD domain, culminating in cleft closure. Similarly, a study of Dictyostelium myosin II using extrinsic probes demonstrated that cleft movement is linked to actin binding (13). Thus, a growing body of evidence suggests that the actin-binding cleft begins to close in strong-binding states of the ATPase cycle even in the absence of actin, and closes to a greater degree upon actin binding.
Although domain movements of the lever arm and actin-binding cleft are now well established in myosin, conformational changes at the active site have proven difficult to observe by crystallography. The active site would be expected to open in the strongly actin-bound states to allow for the exchange of ADP and ATP, and close upon ATP binding to provide a catalytically competent conformation for hydrolysis. Rayment et al. predicted from their original myosin crystal structure that opening of the active site could be triggered by actin-binding cleft closure (14). Further, pivoting the upper-50-kD subdomain around a hinge located near the active site allows for greatly improved fitting of the skeletal myosin crystal structure to cryo-EM reconstructions of the acto-myosin complex (11). Such a pivot would close the actin-binding cleft while opening the active site, as predicted by Rayment et al. (14).
The myosin active site, considered from a perspective closer to the 
-phosphate binding pocket, contains several loop motifs in common with the members of the kinesin and G-protein superfamilies of nucleotide triphosphate-binding proteins (15). Three loops, termed the phosphate-binding loop (P-loop), switch I, and switch II, are thought to play coordinating roles during the hydrolysis cycle. However, aside from these similarities, large structural differences exist between the active sites of G-proteins, kinesins, and myosins. The switch I and switch II loops are exposed in G-proteins and have been shown to participate directly in binding to G-protein targets (16–18); e.g., switch II of the 
-subunit of heterotrimeric G-proteins interacts directly with its ß subunits and switch I of the Rap-1 G-protein interacts with its target Raf. In contrast, in the much larger and more complex myosins, the switches are buried and are not involved in interactions with actin but instead act to coordinate the distal domains that carry out myosin function. Although crystal structures have been obtained for a large variety of G-proteins in all stages of the hydrolysis cycle, in myosin the structural picture is incomplete, as only the weakly actin-bound states have been captured. This may be a consequence of the complexity of myosin active sites relative to those of G-proteins. Even comparing myosins to the closely related kinesins, elements of the active site are thought to act by somewhat different mechanisms, e.g., strong binding of myosin to actin and kinesin to microtubules don't occur in identical nucleotide states. Further, whereas switch I has been shown to close on microtubule binding for kinesins (16), for myosins there is reason to believe that the opposite occurs, as actin binding may trigger opening of switch I (8,11). Thus, despite information about hydrolysis mechanisms in G-proteins and kinesins, the question of how the myosin active site is altered during the ATPase cycle remains open and controversial. Of particular importance is the question of whether structural events at the myosin active site required for enzymatic function can be accommodated solely by rearrangements of the P-loop, switch I, and switch II, or must include more global changes.
In this study, fluorescence resonance energy transfer (FRET) is used to examine distance changes across the active site in nucleotide states that correspond to both strongly and weakly actin-bound states. A chicken smooth-muscle myosin fragment, F344W motor domain essential light chain (MDE), has been genetically engineered to contain a single tryptophan (W344) that acts as the FRET donor in this study, whereas 2'(3')-O-(N-methylanthraniloyl) (mant) fluorescent analogs of ATP and ADP bound to the active site are used as FRET acceptors. The single tryptophan substitution (F344W) is located on a loop between two -helixes that traverse the myosin upper-50-kD subdomain at the front of the active site, whereas the mant moiety of the nucleotide analogs is known to sit in the nucleotide binding pocket (Fig. 1). Measurements made from superimposed crystal structures of Dictyostelium myosin II bound to mant nucleotides (4) and chicken smooth-muscle myosin bound to 
(2) show that the 344W residue is located 
22 Å from the mant moiety, placing it within the useful range of measurement for this FRET donor-acceptor pair. Correlation of FRET efficiency with distance in each nucleotide state has allowed us to monitor a structural rearrangement of the active site that is consistent with the predictions of both Rayment et al. (14) and Holmes et al. (11).
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MATERIALS AND METHODS |
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Solutions
The standard buffer for fluorescence procedures was a 3-[N-morpholino] propanesulfonic acid (MOPS) buffer at pH 7.4 (20 mM MOPS, 20 mM KCl, 1 mM EGTA, 1 mM NaN3, 1 mM dithiothreotol, and 2 mM MgCl2) at room temperature. For protein purification procedures an imidazole buffer at pH 7.4 (10 mM imidazole-HCl, 90 mM NaCl, 1 mM NaN3, and 1 mM dithiothreotol) was used at 4°C.
cDNA construction
Site-directed mutagenesis was performed using a Quik-Change mutagenesis kit (Stratagene, LaJolla, CA) to create a mutant cDNA coding for a chicken smooth-muscle myosin heavy chain containing a single nonendogenous tryptophan at position 344 (highlighted as a space-filling model; Fig. 1). The cDNA used as a template (null-MDE) was a clone coding for the heavy chain containing the motor domain and light-chain binding regions (residues 1–819 kindly provided by Kathleen M. Trybus, University of Vermont) in which the seven endogenous tryptophans of the clone were mutated to phenylalanine (or methionine in the case of Trp-546) as previously described (19). The FLAG epitope (DYKDDDK) was attached to the C-terminus of the cDNA for purification purposes.
Protein expression and purification
The F344W-MDE and tryptophan null-MDE constructs were expressed in baculovirus by coinfecting Sf-9 cells with recombinant baculoviruses containing the mutant heavy chain and the essential light chain. Cells were incubated with baculovirus for 3 days, lysed, fractionated between 30% and 70% saturated ammonium sulfate and dialyzed overnight in imidazole buffer at 4°C as previously described (19). The resulting lysate was purified on an anti-FLAG antibody column, dialyzed overnight into MOPS buffer, and assayed for purity by SDS page on a Coomassie-stained gel (20). Protein concentrations were determined by the method of Edelhoch (21). Fluorescence experiments were performed on 10 such preparations of F344W-MDE. Actin used in actin-activated ATPase assays was purified from acetone powder as previously described (22). The purified actin concentration was determined spectroscopically using an extinction coefficient of 0.63 mg ml–1 cm–1 at 290 nm.
Enzymatic assays
Actin-activated ATPase assays were performed as previously described (23) at 37°C in low ionic strength MOPS buffer (10 mM MOPS, 1 mM EGTA, and 7 mM MgCl2) at pH 7.0 using a range of actin concentrations between 0 and 136 µM. ATPase rates were plotted as a function of actin concentration and fit to the Michaelis-Menten equation using a nonlinear least-squares routine in SigmaPlot 8.0 (SPSS, Chicago, IL) to determine values of Vmax and Km.
Steady-state fluorescence measurements
Steady-state fluorescence measurements of tryptophan fluorescence were obtained on a Quantamaster fluorimeter (Photon Technology International, South Brunswick, NJ) equipped with a 50-W xenon arc lamp excitation source, excitation/emission monochrometers, and a WG-320 cutoff emission filter. Protein samples were excited at 295 nm and tryptophan emission spectra were collected from 305 to 400 nm. Slit widths of 1 nm (excitation) and 5 nm (emission) were used. All spectra were corrected for Raman scatter and background fluorescence by subtraction of an appropriate buffer blank. Spectra were taken at a protein concentration of 1 µM in the standard MOPS buffer (defined above) at room temperature. Spectra of protein plus ATP or ADP were obtained at a saturating nucleotide concentration of 2 mM. Mant ATP and mant ADP were used at nonsaturating concentrations of 15 µM and 10 µM, respectively, to avoid inner-filter effects associated with the mant moiety. F344W-MDE plus mant nucleotide spectra were corrected for nonsaturating concentrations of mant nucleotides using
 | (1) |
for fb, where [Ei] = initial protein concentration [Si] = initial nucleotide concentration, and Kd = the dissociation constant for nucleotide binding to F344W-MDE. Kd values were determined as discussed below. Quantum yields for tryptophan were calculated by comparison to an L-tryptophan standard (
= 0.14) as previously described (24).
Fluorescence lifetime measurements
Lifetime measurements were made using a Photon Technology International (London, Ontario, Canada) Laserstrobe instrument. The excitation source for lifetime experiments was a nitrogen-pumped dye laser. Exciting radiation at 590 nm from the laser was modified to the final 295 nm using a frequency doubler, and fluorescence was collected by a time-delay gated photomultiplier tube. The instrument response function at 295 nm was determined using a dilute solution of nonfat dry milk as a scatterer. The F344W-MDE decays were deconvoluted from the instrument response function and fit to a two-exponential decay using FeliX 32 software provided by Photon Technology International.
Acrylamide quenching experiments
To determine the degree of exposure of the 344W residue to solvent, acrylamide quenching experiments were performed in various nucleotide conditions, including the presence of 2 mM ATP or 2 mM ADP, or in the apo state. The fluorescence values from the 333 nm emission peak in the absence of quencher (F0) were divided by the fluorescence in the presence of quencher (F), plotted against quencher concentration (Q), and fit to the Stern-Volmer equation using linear regression analysis in Kaleidagraph (Synergy Software, Reading, PA):
 | (2) |
0 is the 344W tryptophan lifetime in the absence of quencher, and Ksv is the Stern-Volmer constant (25). Thus, kq was calculated by dividing Ksv by 0, determined by fluorescence lifetime measurements.
Fluorescence resonance energy transfer
The distance between the 344W donor and mant acceptor probes was measured using FRET according to the Förster energy transfer theory (26). We measured the efficiency (E) of FRET by observing the decrease in fluorescence of the 344W donor:
 | (3) |
 | (4) |
R0 was calculated using Eq. 5:
 | (5) |
 | (6) |
is the wavelength, FD is the normalized intensity of the unquenched donor, and 
A is the extinction coefficient of the acceptor. J was determined to be 6.06 x 10–15 M–1 cm3 by numerical integration of our experimentally determined FD and A values at 1-nm intervals for F344W-MDE in the presence of both mant ATP and mant ADP.
Equilibrium nucleotide binding affinity measurements
To determine F344W-MDE-mant ADP affinity by steady-state fluorescence, the protein was titrated with increasing concentrations of nucleotide in a series and the decrease in tryptophan fluorescence emission at 333 nm was monitored in the fluorimeter after excitation at 295 nm. These fluorescence values were corrected for inner-filter and dilution effects, normalized to the fluorescence value obtained for nucleotide-free protein and subtracted from 1. Plotting (1 – normalized fluorescence) versus [mant nucleotide] produces a rectangular hyperbola which approaches a maximum binding value (Bmax) asymptotically:
 | (7) |
The resulting plots were used to determine dissociation constant (Kd) values (equal to the nucleotide concentration at which 0.5·Bmax occurs) by fitting Eq. 7 to the data using SigmaPlot 8.0 (SPSS Corp., Chicago, IL). The affinity of the F344W-MDE protein for mant ATP was determined similarly by exciting at 295 nm and after both the tryptophan emission at 333 nm and the mant ATP fluorescence emission at 440 nm (in separate experiments). Unlike the case for mant ADP, these titrations could not be performed in a continuous series due to ATP hydrolysis during the experiments. Therefore, each specific concentration of mant ATP was added to protein and fluorescence at 333 nm was monitored in the fluorimeter as a function of time.
Transient kinetic analysis of mant-ADP binding
The affinity of F344W-MDE for mant ADP was also determined by making stopped-flow measurements of the binding and release rates of mant ADP, where Kd = koff/kon. To measure the apparent second-order binding constant (kon) for mant ADP, 2 µM F344W-MDE in one syringe was rapidly mixed with various concentrations of mant ADP (0.3–5 µM) in the second syringe. To determine the rate of mant ADP release (koff) from F344W-MDE, chase experiments were performed in which a complex of mant ADP (10 µM) bound to 2 µM F344W-MDE in one syringe was rapidly mixed with an excess of ATP (500 µM) in the second syringe. The rate of mant ADP binding or release was determined by following the change in mant ADP fluorescence above 420 nm with a 420 cutoff filter after excitation at 295 nm (5-nm bandpass). All transient kinetic experiments were done using a BioLogic µSFM-20 stopped-flow instrument (Claix, France) with a dead time of 2.1 ms under the flow-rate conditions used. The resulting data were fit using nonlinear regression analysis in Kaleidagraph (Synergy Software, Reading, Pa.). Experiments were performed in the MOPS buffer as described above for steady-state fluorescence experiments at 25°C.
Protein structural comparisons
The three-dimensional structures of myosin isoforms were superimposed and visualized in Swiss-PdbViewer (GlaxoSmithKline, Middlesex, UK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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,27). Thus, the F344W-MDE protein behaves much like other single-tryptophan MDE mutant proteins and the wild-type chicken smooth-muscle MDE protein in terms of its ability to hydrolyze ATP in an efficient and actin-dependent manner.
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). However, addition of saturating ATP leads to a more complex situation as hydrolysis of the triphosphate can occur and thus myosin will be found in a mixture of nucleotide states with the percentage of myosin in each state being determined by the rate constants for the protein in the ATPase cycle, as discussed below. No spectral shift of the F344W-MDE emission maxima of 333 nm was seen in the steady-state spectra in the presence of ATP or ADP; the implications of this are also discussed below.
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In addition to the strongly fluorescent 344W residue, the F344W-MDE contains 28 endogenous weakly fluorescent tyrosines. Although our experiments were done at an excitation wavelength of 295 nm, which should produce little or no tyrosine excitation, we controlled for the contribution of tyrosine fluorescence by subtracting emission spectra obtained under identical experimental conditions of a "null-MDE" mutant from our F344W-MDE emission spectra. The null-MDE is identical to the F344W-MDE except for the presence of a phenylalanine at position 344 and thus contains no tryptophan residues. Our subtractions of null-MDE spectra from F344W-MDE spectra confirm that the tyrosines in our MDE construct make no contribution to our FRET results (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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) and the pivoting of the upper-50-kD subdomain used by Holmes et al. (11) to fit myosin ADP structures to cryo-EM reconstructions. Although FRET results in general should not be misinterpreted as giving accurate determinations of distance, our FRET-measured distance change of 8.5 Å is of sufficient magnitude and certainty to show a definite distance change between the 344W residue and mant moiety in the presence of ADP as compared to the presence of ATP. The most straightforward explanation of this opening is movement of the upper-50-kD subdomain. However, it is also possible that our FRET results reflect a more local rearrangement of the short loop that contains the 344W residue, and/or that the mant moiety is repositioned during the ATPase cycle, contributing to the observed distance change. Even in the case in which a combination of nucleotide repositioning and local 344 loop movement causes the opening between the 344W residue and mant moiety in the ADP state, our results still demonstrate a rearrangement of the core active site. Thus, our results provide the first evidence in solution consistent with a movement of upper-50-kD subdomain that opens the myosin active site in a strongly actin-bound state of the ATPase cycle. Such an opening may be necessary to coordinate nucleotide hydrolysis to actin binding. We therefore propose a model of myosin domain coordination in which movement of the 50 kD cleft during actin binding is communicated to the active site through a pivoting of the upper-50-kD subdomain as the 50-kD cleft closes on actin, effectively opening the active site and increasing the rate of product release. Changes in the interactions and positions of switch I and switch II upon product release would then be communicated to the converter domain through the switch II helix, triggering the powerstroke only after myosin has entered the strongly bound state. As the power stroke is almost certainly timed to occur when myosin is strongly bound to actin, the necessity of such a mechanism to link product release to actin binding is understandable.
Interpretation of our steady-state results using unlabeled nucleotides
Our steady-state results using unlabeled ATP and ADP suggest that structural changes occur during the ATPase cycle that place the 344W residue in a different environment in the apo, ADP, and ATP states. As no spectral shift was seen in the presence of either nucleotide, it is likely that the attenuation of fluorescence was not the result of a large change of exposure of 344W to solvent, but instead probably reflects closer proximity of 344W to a charged protein side chain capable of altering the fluorescence emitted by 344W. Research on mechanisms leading to fluorescence quenching of tryptophans in proteins suggests that charged residues can influence quantum yield by stabilizing a charge-transfer state in which the peptide bonds accept the excited electron from tryptophan (29). Inspection of the chicken smooth-muscle myosin amino acid structure shows several such residues, including a string of three glutamic acid residues located at positions 346–348, which could provide such a function. This conclusion is supported by our quenching results, which showed nearly identical bimolecular quenching constants for the apo, ADP, and ATP states, respectively. Regardless of the underlying basis for the differential quenching observed in the ADP and ATP states, we have obtained the appropriate donor-only signals in each state to use in determinations of FRET efficiency in the presence of mant nucleotide.
Technical issues and caveats
Because the mant moiety used as our FRET acceptor absorbs strongly at both the excitation wavelength used in our experiments (295 nm) and at the emission maxima of the 344W tryptophan (333 nm), accurately correcting for the substantial inner-filter effects obtained at high mant nucleotide concentrations represented a difficulty. We avoided this inaccuracy by performing our fluorescence experiments at nonsaturating mant nucleotide concentrations of 10 µM mant ADP and 15 µM mant ATP. Therefore our determinations of FRET efficiency were dependent on knowing the fraction of protein bound to mant nucleotide, which we calculated based on the affinities of mant nucleotides for F344W-MDE. The excellent agreement between the two very different methods we used to determine the affinity of F344W-MDE for mant ADP affinity gives us confidence in our correction for the fraction of mant ADP bound to F344W-MDE in the FRET experiments. In the case of mant ATP we also had excellent agreement between two different measurements of Kd. Due to the hydrolysis of mant ATP during each titration it was not possible to titrate with very low concentrations of mant ATP. However, the concentration of mant ATP used in the FRET experiments (15 µM) relative to the saturation point observed in our steady-state titrations (>6 µM) strongly suggests that F344W-MDE was indeed almost completely saturated.
The Kd values obtained for F344W-MDE (mant ATP, 0.8 µM; mant ADP, 8.2 µM) are in close agreement with values we obtained for the null-MDE (mant ATP, 0.5 µM; mant ADP, 8 µM) and Kd values determined for W29-MDE, another single tryptophan MDE mutant protein constructed in our lab (mant ATP, 1 µM; mant ADP, 7 µM; data not shown). Working with smooth-muscle wild-type S1 myosin II, Cremo and Geeves (28) obtained a Kd of 0.075 µM for mant ADP and 16-fold higher Kd for ADP of 1.2 µM. However, Kd values for various myosin II S1 proteins for ADP range widely, from 0.33 µM for cardiac S1 (30) to 37 µM for Dictyostelium myosin II (31), and thus it is not surprising that the single tryptophan-MDE mutant smooth-muscle myosin varies from the wild-type S1 in its nucleotide affinities.
There are a number of caveats that must be addressed regarding interpretation of our FRET data. When F344W-MDE is combined with ATP or mant ATP, we would expect myosin to be found in a mixture of intermediate states, with the weakly actin-bound M·ATP and M·ADP Pi species predominating. Rate constants previously determined for the elemental steps of ATP hydrolysis by smooth-muscle myosin II suggest that the M·ADP Pi species should be the most populated state, as the forward hydrolysis step is favored over the reverse hydrolysis step and phosphate release is rate-limiting (32). Our results in the presence of mant ATP therefore most likely represent a mixture of weakly actin-bound states of the myosin ATPase cycle.
Because our FRET study was done on a population of molecules in solution, the FRET distances reflect average distances between 344W and the mant moiety for the population. As it is a near certainty that myosin interconverts between a wide range of conformations in each nucleotide state (i.e., the myosin energy landscape is populated by many variants in each nucleotide state of the ATPase cycle), our FRET study cannot distinguish these conformations and dynamic motions within them. In fact, conformations that are relatively rare and thus contribute little to FRET may be highly important in terms of experiencing interactions that drive myosin through the ATPase cycle. However, this caveat is true of any FRET solution study and does not decrease the value of our results.
Finally, in calculating R0 between the 344W and mant nucleotides we made the assumption that the orientation factor, K2, is equal to 2/3. This implies that the 344W-mant nucleotide FRET donor-acceptor pair are free to rotate and thus rotational diffusion between the probes will occur after excitation but before energy transfer takes place. Although the possible range of K2 values is 0–4, when the 2/3 assumption is made the FRET distance cannot be in error by >35% (33). For example, if K2 was in reality 0.1 in the presence of both mant ADP and mant ATP, the distance change between the weakly and strongly actin-bound states of the ATPase cycle decreases to only 6.2 Å, still a significant difference. An increase in K2 would only serve to increase the observed difference in distance between these states. Thus it is clear that variation in K2 from the assumed 2/3 value would not significantly affect our basic conclusion that there is a large-scale 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.
Comparison to available crystal structure data
Superimposition of mant nucleotide coordinates from Dictyostelium myosin II (4) on the chicken smooth-muscle myosin MDE structure (2) showed a distance between the mant moiety and 344W of 22.4 Å, in very good agreement with our FRET-determined distance. Distance measurements from both the P-loop and the ribose ring of the bound nucleotide to F344 or its equivalent show that little variation in these distances occurs among nucleotide-bound myosin II structures, most likely because these structures capture weak actin-binding states with switch I closed and nucleotide tightly bound.
Recently Coureux et al. (9,34) obtained chicken myosin V structures in the absence of nucleotide or soaked with ADP that show switch I in an open position, as well as an ADP.BeFx structure that resembles previous myosin II structures with switch I closed. Another recent apo structure for Dictyostelium myosin II (8), is in close agreement with the myosin V apo structure. By comparison with the myosin II nucleotide-bound structures the P-loop has moved closer to the 344 position in the myosin V ADP and apo structures (Fig. 6). Thus, the conformational state observed for smooth-muscle myosin in the presence of ADP in this study, in which 344W has moved further away from the nucleotide, is likely to be unique compared to the ADP or apo structures seen in myosin V. Although the myosin V-ADP structure does capture switch I in an open position, the authors state that this structure likely corresponds to a weakly ADP-bound state of myosin V (34). In contrast, the ADP state captured in our study is likely a tightly ADP-bound state. Rosenfeld et al. (35) demonstrated that, unlike skeletal and Dictyostelium myosin II, smooth-muscle myosin is capable of adopting a unique strong actin-binding, tightly ADP-bound state, i.e., actin and ADP binding can be loosely coupled in smooth-muscle myosin. They argued that the tight affinity for ADP in this state may be mediated by loop 1, which would explain the fact that the active site in our smooth-muscle myosin construct is open and yet binds ADP relatively tightly. A unique strongly bound ADP state has also been observed in smooth-muscle myosin by Whittaker et al. (36), who used cryo-EM and helical image reconstruction to demonstrate that ADP release generates an additional tilt of 23° of the regulatory domain in chicken gizzard smooth-muscle myosin not seen in skeletal and Dictyostelium myosin II. Therefore, our smooth-muscle ADP FRET result may capture a myosin state that does not exist in skeletal and Dictyostelium myosin II isoforms.
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), in which rigid rotation of the upper-50-kD subdomain upon actin binding serves to close the actin-binding cleft and open switch I at the active site. The presence of ADP may stabilize the active site in a more open conformation and/or the actin-binding cleft in a more closed conformation in the absence of actin. Another intriguing possibility is that a more open active site in the ADP state is physiologically relevant in terms of product release.
Future work
The ability of the 344W residue to indicate movement of the upper-50-kD subdomain, which correlates to changes between weakly and strongly actin-bound states, provides a useful fluorescent signal for use in future kinetics studies to correlate structural rearrangements of the myosin active site to the biochemical events of the ATPase cycle. As well, such studies can provide a means to correlate active-site movement with motions of other myosin domains that must be coordinated to produce directed force and motion during muscle contraction and other forms of cell motility.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant to C.L.B. from the National Institutes of Health, HL 63798.
Submitted on January 19, 2005; accepted for publication May 25, 2005.
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REFERENCES |
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) or (B) ATP (+) and mant ATP (
). Spectra are corrected for the fraction of F344W-MDE not bound to nucleotide. Quenching of 344W fluorescence is observed in the presence of each nucleotide: ADP, 81% apo; mant ADP, 76% apo; ATP, 76% apo; mant ATP, 58% apo. Quenching of fluorescence relative to the apo state in the presence of nucleotides lacking the mant moiety is due to changes in the 344W environment. Additional loss of intensity in the presence of mant nucleotides (e.g., mant ATP compared to ATP or mant ADP compared to ADP) is due directly to FRET between 344W and the mant moiety at the active site.
), and ATP (
) conditions. F0/F was plotted against [acrylamide] and fit to the Stern-Volmer equation (
