Repriming the actomyosin crossbridge cycle

Walter Steffen and John Sleep*

Randall Centre, King’s College, London SE1 1UL, United Kingdom

Edited by Edwin W. Taylor, University of Chicago, Chicago, IL, and approved June 8, 2004 (received for review January 12, 2004)

The central features of the mechanical cycle that drives the con- traction of muscle are two translational steps: the working stroke, whereby an attached crossbridge moves relative to the actin filament, and the repriming step, in which the crossbridge returns to its original orientation. Although the mechanism of the first of these is understood in some detail, that of the second has received less attention. Here, we show that repriming occurs after detachment of the crossbridge from the actin, rather than inter- vening between two actomyosin states with ATP bound [Eisen- berg, E. & Greene, L. E. (1980) Annu. Rev. Physiol. 42, 293–309]. To discriminate between these two models we investigated the single-molecule mechanics of the myosin–actin interaction in the presence of ATP analogues such as GTP, for which the hydrolytic step itself limits the actomyosin GTPase rate to a much lower rate than for ATP. The lifetimes of bound states was proportional to 1͞[GTP], indicating that during the bound period myosin was in the actomyosin rigor configuration. Moreover, despite the very low actomyosin GTPase, the rate of actin binding and formation of the rigor state was higher than with ATP; it follows that most inter- Fig. 1. Models of the actomyosin crossbridge cycle. (A) The L-T alignment of actions with actin result in the release of GTP and not of the biochemical states with the crossbridge cycle. Repriming occurs between products, GDP and phosphate. There was no significant movement dissociated states (M⅐T 3 M⅐ D⅐ P). (B) Eisenberg and Greene’s proposal in of the actin during this interaction, so repriming must occur while which the weak states (M⅐T and M⅐D⅐P) bind to actin in the same preforce myosin is dissociated, as in the original Lymn–Taylor scheme conformation. Repriming occurs between attached states (AM⅐T 3 AM⅐T). [Lymn, R. W. & Taylor, E. W. (1971) Biochemistry 10, 4617–4624]. Italics indicate postforce states.

he mechanical events responsible for muscle contraction are working stroke, both take place with myosin attached to actin. coupled to the actomyosin ATPase cycle. According to the T However, the kinetic asymmetry accounts for this point: the basic scheme of Lymn and Taylor (L-T) (1) (Fig. 1A), in the ⅐ weakly bound, negative-force-generating AM T90 state very rap- relaxed state (crossbridge 90° to the filament axes) the myosin Ϫ idly dissociates [Ϸ5,000 s 1 (7)], whereas the positive-force- (M) is bound to ADP (D) and phosphate (P). The binding of generating AM⅐D state is more long-lived by 2 orders of mag- actin (A) to M⅐D⅐P leads to the working stroke, accompanied 90 nitude (5). by product release and the formation of the rigor AM45 state. ATP (T) binds to AM to form AM⅐T, from which actin rapidly Our purpose here was to determine whether the L-T scheme dissociates. Hydrolysis then takes place while myosin is dissoci- correctly locates the repriming step, or whether it accords with ated from actin, and the mechanical repriming of the cross- the alternative proposal of Eisenberg and Greene (5). To bridge, which positions it for the start of the next cycle, is distinguish between the two models we needed to establish associated with this step. Further research by Eisenberg and whether or not repriming takes place while myosin is associated collaborators (2) added important details to the scheme. The with actin, that is, in the part of the cycle between AM and ϩ ⅐ concentration of Ca2 regulates the binding to actin of the AM T. With sufficiently good time resolution this question could so-called ‘‘strong binding’’ set of myosin states (M and M⅐D) but be answered by studying interactions with ATP (see Fig. 2 a and Ϸ has little effect on the binding of the ‘‘weak’’ states (M⅐T and b) but at present event detection is currently limited to 10 ms M⅐D⅐P). This finding suggested that these two sets of states bind and the direct approach is not viable because the answer is to actin in different conformations (2), from which it was natural hidden in the gray, temporally unresolved bands (Fig. 2 a and b). to infer that the transition between these conformations might The difference in positions during the free and bound periods is correspond to the working stroke. Moreover, the free energy a measure of the displacement of the AM state with respect to change between M⅐T and M⅐D⅐P was very small (3), and the two the reference free dumbbell position. For this to be a valid complexes bound to actin with similar affinities (4), at least 2 measure of the working stroke, all of the transitions between the orders of magnitude weaker than that of the strong states. The dissociated state (M⅐T 7 M⅐D⅐P) and the rigor state (AM) must actin association constant appeared to be a marker for the be via product dissociation. However, it has been reported (6, 8, inferred two conformational states. In this case, as argued by 9) that during acto-Sl ATPase Ϸ10% of interactions of the Eisenberg and Greene (5), the logical position for the repriming dissociated myosin states (M⅐T 7 M⅐D⅐P) with actin correspond ⅐ step would be the transition from a strongly bound AM T45 to a to ATP release. If this subset of interactions could be identified, ⅐ weakly bound AM T90 state (see Fig. 1B) and not the hydrolysis they would report on the displacement between the reference step as in the original L-T model. This proposal had an attractive symmetry that was further evidenced by the observation that actin is almost as effective in releasing ATP from M⅐Tasin This paper was submitted directly (Track II) to the PNAS office. releasing the products from M⅐D⅐P (6). At first sight the model Abbreviation: L-T, Lymn and Taylor. appears to suffer from the difficulty that the repriming (effec- *To whom correspondence should be addressed. E-mail: [email protected]. tively a reversal of the working stroke), as well as the forward © 2004 by The National Academy of Sciences of the USA

12904–12909 ͉ PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400227101 Downloaded by guest on September 26, 2021 Fig. 3. Use of a rotating microscope slide to move both traps to the left by the same extent. In our standard configuration, using a 1-mm thick slide, the displacement at the stage is Ϸ20 nm per degree of rotation

is submillisecond and quite beyond the time resolution of detecting events in the trap. Although the dissociation rates are slower for the analogues [380 sϪ1,ATP␥S (10); 580 sϪ1 at 10°C, GTP (11)], they are still too fast to allow detection of the ternary complex (AM⅐NTP). There are thus two classes of events associated with M⅐NTP binding to actin. The major class is a binding followed by immediate dissociation and goes undetected. The minor class is binding followed by dissociation of NTP and the formation of AM, and it is this class, perhaps only 1% of the total interactions, which we characterize in this article. Exactly equivalent considerations apply when measuring the working stroke in the presence of ATP. Materials and Methods Biochemistry. The biochemical methods have been described (12). Fig. 2. Predictions for interactions based on the L-T and Eisenberg–Greene In outline, cover slips were coated with a suspension of 1.5 ␮m models. (a and b) In the presence of ATP. An arrowhead marks the working silicia beads in 0.05% nitrocellulose followed by BSA. Rabbit stroke and an arrow marks the repriming event in the two models. For the myosin S1 containing a biotinylated regulatory light chain bound purposes of clarity, the duration of the transitions between detached and fairly selectively to this surface. It was applied at a similar rigor states (gray bands) have been exaggerated (expected to be submillisec- Ϸ ␮ ͞ ond). With the present technology, the details within the gray band cannot be concentration ( 1 g ml) for experiments with ATP and for the resolved. (c and d) In the presence of GTP, showing the way in which the analogues. Dumbbells were formed from biotinylated actin held location of the repriming step affects the position of the dumbbell through- between neutravidin-coated beads. out the bound period. BIOPHYSICS Optical Trapping. The optical trap apparatus has been described (12). Because the dumbbell does not rotate about the axis of the position (dissociated state) and AM accessed via ATP release actin filament, to obtain the working stroke it is necessary to and allow assignment of the location of the repriming step. move the dumbbell past the myosin molecule by an integral Ϸ Unfortunately, the amplitude of the brownian noise (SD 10 number of actin repeats (n ϫ 37 nm) past the myosin during the nm) relative to that of the working stroke prevents separation, period of data collection (12). We find that if the traps are moved which results in the interactions observed during ATP hydrolysis by the application of a linear voltage ramp to the acousto-optic Ϸ being a mixture of the product release events of interest ( 90%) deflectors, the resultant motion of the trapped beads can show Ϸ and the contaminant ATP release events ( 10%). This point will signs of steps, perhaps as a consequence of standing sound waves be considered in more detail later. in the AOM crystals. The effect is variable in amplitude, is not As the ATP release events cannot be separated, is it possible always easy to control, and could result in the recording of to increase their proportion so as to be dominant? We have artefactual periodicities. To achieve a uniform motion of the investigated the properties of a class of ATP analogues (e.g., trapped beads a stepper motor-driven micrometer was used to GTP, ITP, and ATP␥S) known to have favorable kinetic prop- rotate by a few degrees a microscope slide placed in the trap- erties. In the case of ATP, the rate-limiting step of the reaction ping beam (Fig. 3). In common with most trap setups, ours has cycle is product release, and the dominant steady-state inter- a beam-expanding telescope to match the diameter of the laser mediate is M⅐D⅐P. The major action of actin is to bind to this state beam to the back aperture of the objective. Lateral movement of and accelerate product release, thus circumventing the slow the beam between the two lenses of the telescope moves the rate-limiting step. However, as noted above, a relatively minor focus of the laser beam. The movements of both beams are equal, activity of actin is to bind to M⅐T and accelerate ATP release. and for small movements they linearly depend on the angle of The ATP analogues we have investigated share the property that rotation of the microscope slide. Fig. 4 is a plot of the SD of the the rate-limiting step of myosin NTPase is the hydrolytic step. bead position against mean bead position (13) for 5-ms time The resultant change in the dominant intermediate from M⅐D⅐P slices and the uniform population of the high-variance cloud, to M⅐NTP offers the prospect of the main interaction of actin representing time slices in which the dumbbell is not interacting being to release NTP. This point has been confirmed, both by the confirms the linearity of bead motion. The low-variance patches trap experiments reported here and independently by biochem- represent interactions with individual actin monomers within ical methods.† each target zone (12). The rate of dissociation of actin from AM⅐ATP is Ϸ5,000 sϪ1 The positions of the trapped beads were recorded at 10 kHz [extrapolated up to 20°C (5)], and thus the lifetime of AM⅐NTP for periods of 100 s. Interactions were detected by the reduction in variance of bead position during bound periods (14). The variance record was analyzed in terms of a simple two-state (low †White, H. D., Schmitz, S., Belknap, B. & Veigel, C. (2004) Biophys. J. 86, 574 (abstr). and high variance) model by using a hidden Markov method as

Steffen and Sleep PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 ͉ 12905 Downloaded by guest on September 26, 2021 Fig. 4. Interactions in the presence of MgATP. (a) Histogram showing position of bound events during a period in which a myosin was allowed to explore two target zones by moving the dumbbell past the myosin at a uniform velocity. (b) Plot of the SD of bead position against mean bead position for 5-ms time slices of a 100-s period of data collection. Below is an Fig. 5. Interactions in the presence of MgITP. (a) Four-second record of head actin filament, scaled to the x axis: the effects of the target zones and the position with 100 ␮m ITP. (b and c) Histograms showing the positions of bound individual actin monomers are clearly evident. events during a period in which the dumbbell was moved past the myosin at a uniform velocity (b, in the presence of ATP␥S; c, in the presence of GTP). The rate of success in achieving this level of resolution was similar (Ϸ50%) to that described (15). This method detects binding events, finds the found for ATP. Failure to observe interactions at this resolution was almost optimum assignments for the rates to and from the low-variance certainly caused by either unfavorable attachment of the Sl to the surface, state (conventionally termed f and g), and determines the high-compliance links between actin and the trapped beads, or occasionally average variance levels for the two states. From these levels and poor operation of the feedback loop used to keep the myosin-coated bead in the covariance of the trapped beads during bound periods, it also a constant position with respect to the trapped beads. With good compo- nents, interactions could be assigned to individual monomers for essentially calculates the compliances of the myosin and the links between all dumbbells tested and most . the two trapped beads and the actin filament. The assumption that the behavior can be described by the two rate constants, f and g, is justified by the results (see Figs. 6 and 7). It should be havior was also observed when ATP was replaced by any of the made clear that, in common with almost all previously reported three different substrates, which provides good evidence that actomyosin single-molecule experiments, we are recording interactions are with a single myosin head. Two examples event-averaged working strokes, that is the mean value of the (ATP␥S and GTP) are shown in Fig. 5. A kinetic argument can average displacements during each recognized actomyosin in- be used to confirm that the low-variance state observed is the teraction. If the displacements during all of the bound events rigor state AM for both the analogues and ATP. If the only were averaged so as to give the time-averaged working stroke, low-variance state is AM then a plot of the number of events then, in the absence of an energy source, the answer must be versus event duration will as observed correspond to a single zero. This thermodynamic constraint does not apply to the event-averaged working stroke. The relation of the throw of the crossbridge (commonly termed h) to the observed, event- averaged, working stroke is model dependent, but in the absence of specific information we assume that the relation will be the same for the binding of M⅐GTP as for M⅐ADP⅐Pi. This notion is reasonable, as if a displacement did result from M⅐GTP binding, then the reaction (involving opening of the switch 2 element) would be of an analogous kind to the conventional working stroke. Results The ATP analogues, GTP, ITP, and ATP␥S, have in general been used to investigate weak interactions (16), but the trap results immediately revealed the presence of strongly bound states (Fig. 5a). The traces are very similar in nature to those observed with ATP. The ratio of the variances of bead position with the actin free and bound was typically Ͼ12, indicating that the attached state was mechanically similar to that of the rigor state previously characterized (15, 17). The stiffness of the combined traps was 0.04 pN͞nm, and the myosin stiffness was Ϸ Ϸ ͞ Fig. 6. The relative frequency of events plotted against event duration. ATP typically 50 times greater ( 2pN nm). The bound states of all (E), ATP␥S(ᮀ), and GTP ({). Concentrations, fitted rates, and total numbers of three ATP analogues studied were found to share this property. events were, respectively, 40 ␮M, 122 sϪ1, and 4,013; 500 ␮M, 108 sϪ1, and As described in Materials and Methods, a characteristic feature of 18,963; and 200 ␮M, 56 sϪ1, and 13,811. At the relatively high nucleotide interactions in the presence of ATP is that they can be assigned concentrations featured in this plot the rates fitted in this manner are some- to specific actin monomers within target zones (12). This be- what higher than given by the global fit of the hidden Markov method.

12906 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400227101 Steffen and Sleep Downloaded by guest on September 26, 2021 Fig. 7. Dependence of the rate of dissociation on [ATP␥S], [GTP], and [ITP]. The GTP and ATP␥S experiments were done with our normal buffer A of 25 mM KCl, 25 mM Hepes, and 4 mM MgCl2, and the ITP was done in the presence of an additional 25 mM KCl to reduce the rate of binding to actin. The fitted lines have been constrained to a zero rate at zero [nucleotide] although there Fig. 8. Histograms of the mean positions during each bound period. (a)50 is evidence of slightly anomalous behavior at very low concentrations. ␮M GTP. (b) 100 ␮M ATP␥S.

exponential. For ATP, the good fit (see Fig. 6) indicates that the release. The polarity of the actin filament determines whether lifetime of any low-variance state with bound products is shorter any displacement will be to the left or right, but for the ATP than can be detected by using the variance method of analysis analogues used here the observed displacement was too small to (less than Ϸ10 ms). The plots for GTP and ATP␥S are also well fitted by single exponentials, as expected from the high maxi- establish the polarity and it was not independently assayed. The mum rate of dissociation at saturating concentrations of these best estimates of the displacement were obtained by taking as analogues; the occupancy of a low-compliance AM⅐NTP state is large as possible a data set with a single dumbbell, which was not evident. The duration of events is thus controlled solely by allowed to interact with as many myosin subfragment-1 mole- the rate of nucleotide binding, and Fig. 7 gives a plot of the rates cules in succession as possible. Histograms showing the distri- of dissociation as a function of nucleotide concentration. The bution of bound states for the best individual dumbbell when using GTP and ATP␥S are shown in Fig. 8. The mean values of observed linearity over the experimentally accessible range Ϯ Ϯ BIOPHYSICS confirms that AM is the only significant low-variance state. The these distributions were 0.4 0.7 and 0.0 0.2 nm, respectively. apparent second-order rate constants 2.8 ϫ 105, 1.4 ϫ 105, and An estimate of the upper limit was also derived by taking the 1.2 ϫ 105 MϪ1⅐sϪ1 for GTP, ITP, and ATP␥S, respectively, are mean of the absolute value of the estimates of the displacements comparable to solution values for acto-Sl (10, 11). We expect the from a number of different dumbbells. Table 1 summarizes ␥ second-order rate constants deduced from the trap experiments measurements of the displacements for ITP, GTP, and ATP S, to be very similar to solution values but there is no such clear which are all zero within experimental error and at least for these relation between the second-order rate of actin binding to M⅐D⅐P substrates, the pathway involving repriming before dissociation Ͻ (and M⅐T) and the rate of occurrence of myosin-binding events, accounts for 10% of the total flux. which critically depends on the exact positioning of the dumbbell Discussion with respect to the interacting Sl molecule. However, the effect of these less controllable parameters will average out over a Single-molecule mechanical methods have been used to explore number of experiments, and for all three alternative substrates, the repriming step of the crossbridge cycle by analyzing the the rate is clearly a few times higher than for ATP (see Table 1). partial reaction of the actomyosin cycle between A ϩ M⅐T 3 Most of the ITP experiments were done at a slightly higher ionic AM⅐T 3 AM ϩ T. For this purpose we used three ATP strength than customary (additional 25 mM KCl) because oth- analogues for which the dominant steady-state intermediate erwise rapid rebinding events vitiated the operation of the during NTPase activity is M⅐NTP. The duration of bound events analysis program. The focus of our experiments was to measure was found to be proportional to 1͞[ATP analogue] in the the shift between the mean position of the dumbbell while bound experimentally accessible concentration range, as expected if the and free (14). We will use the term displacement to avoid species giving the stiff, low-noise interaction were the rigor AM confusion with the working stroke associated with product state. Actin is a very much poorer activator of the myosin

Table 1. Working strokes for different myosin nucleotide complexes Nucleotide, ␮M

ATP, 10 ITP, 50 ITP GTP, 200 GTP ATP␥S, 200 ATP␥S

Apparent working stroke 5.4 0.5 Ϯ 0.8 nm 0.8 Ϯ 0.6 nm 0.4 Ϯ 0.7 nm 0.6 Ϯ 0.7 nm 0.0 Ϯ 0.2 nm 0.2 Ϯ 0.3 nm No. of events 5,580 4,099 27,028 8,399 17,507 9,105 17,252 No. of records 18 ϫ 100s 13 ϫ 100s 44 ϫ 100s 15 ϫ 100s 49 ϫ 100s 10 ϫ 100s 38 ϫ 100s Rate MϪ1⅐␮sϪ1 2 0.16 0.24 0.4 On rate 1ϫ 5.2 ϫ 4 ϫ 4 ϫ No. of myosins 5 3 15 3 12 3 10

Steffen and Sleep PNAS ͉ August 31, 2004 ͉ vol. 101 ͉ no. 35 ͉ 12907 Downloaded by guest on September 26, 2021 characteristic element of P-loop proteins) (19–21). The central features of the crossbridge cycle, the working stroke, and the antagonistic behavior of ATP and actin binding to myosin thus finally have a plausible structural interpretation. In retrospect it has become apparent that Eisenberg and Greene’s weak and strong states may well have some relation to states with closed and open switch 1 elements rather than, as they originally conjectured, with the working stroke, that is, with the switch 2 element. The full mechanism may be rather more complicated but this simple scheme accounts for our results and is convenient Fig. 9. Interaction of myosin intermediate states with actin. (a) Possible for the purposes of discussion. interactions with actin. Subscripts c and o refer to states with closed and open We have previously reported that the binding of the switch 2 switch 2 elements. The open, postworking stroke states are in the front plane open states, M⅐PPi, M⅐ADP, and M, to actin, is in no case and are in italics. (b) The main pathways of interaction: the M⅐D⅐P must interact associated with a detectable working stroke. The only state ⅐ via the closed form to give a working stroke, whereas M T must do so via the observed to give a working stroke was the closed state M⅐D⅐P open form despite the existence of an approximately equal concentration of (22), and this set of observations fits readily into the proposed closed form. structural model, in which a working stroke results exclusively from the opening of switch 2 and not from other events NTPase for these analogues than for ATP. Our observation that associated with actin binding. An important aspect of the the interaction rate is significantly higher with GTP, ITP, and structural model is that the nucleotide pocket must close before ATP␥S than with ATP (see Table 1) provides compelling the is in the appropriate conformation to catalyze evidence that almost all interactions are caused by actin binding hydrolysis. This proposition has been substantiated by fluores- ⅐ cence studies (23, 24), which have given evidence for both open to M NTP, leading to dissociation of NTP and the formation of ⅐ the rigor AM state. It has previously been shown that the and closed forms of M T. Our results can now be interpreted in ⅐ ⅐ ⅐ terms of the repriming step corresponding to the transition addition of actin to an equilibrium mixture of M T and M D P ⅐ ⅐ ⅐ ⅐ releases Ϸ10% of the bound nucleotide as ATP (6). A similar between Mo T and Mc T (as opposed to AMo T and AMc T for study showed that in the case of GTP Ͼ90% of the bound the Eisenberg–Greene proposal). Is it safe to extrapolate conclusions regarding repriming from nucleotide is released as GTP,† which confirms our conclusions these ATP analogues to ATP itself? A potential weakness in the that the events we observe are associated mainly with GTP argument arises from the possibility that hydrolysis is rate release. The contribution of AM⅐NTP states to the low-noise limiting for the GTP-like analogues because the open to closed periods is below the level of detection because the rate of actin transition is much less favorable than for ATP. This process dissociation (AM⅐NTP 3 A ϩ M⅐NTP) is greater than the time would result in a reduction in observed rate of hydrolysis and resolution of our method of analysis. populate the Mo⅐T state at the expense of Mc⅐T, thus favoring The state AM45 is a postforce 45° state, and we must assume ⅐ formation of AM without an observable displacement. Fortu- that the first state upon ATP binding is AM45 T. The reverse ⅐ nately, it has been reported (25) that the equilibrium between the path from M TtoAM45 must pass through this state. If the ⅐ ⅐ open and closed forms of M NTP for the analogues are all quite preceding state is M T45, no displacement will be observed (L-T), Ϸ Յ Յ Յ ⅐ comparable to that for ATP ( 0.4, 0.3, 0.7, and 1.7 for whereas if the preceding state is AM90 T, a displacement will be ATP, 〈⌻P␥S, GTP, and ITP, respectively). A second kinetic observed (Eisenberg–Greene). The relative fluxes from these argument favors dissociation before repriming for ATP. The two sources depend on all of the rates linking the four states: ⅐ ⅐ ⅐ ⅐ rigor state AM is an open state (AMo) and the first ATP state AM45 T, AM90 T, M45 T, and M90 T. We observe no displace- must be AMo⅐T. The first-order rate of actin dissociation from ment upon M⅐GTP binding to actin and forming AM and thus ⅐ Ͼ Ϫ1 ⅐ AM Tat25°Cis 5,000 s (7), which is probably significantly conclude that M GTP must bind to actin in the same mechanical faster than the competing repriming step (AMo⅐T 3 AMc⅐T). conformation as the rigor state (45°). Consequently, the re- The latter rate has not been measured but the rate of the priming step of the contractile cycle (90° to 45° transition) must equivalent step in the absence of actin is Ϸ300 sϪ1 (23, 25) and occur while myosin is dissociated from actin (L-T). The results the overall rate of the composite hydrolysis step (i.e., open to 〈⌻ ␥ for ITP and P S were very similar. The observation of a null closed and hydrolysis itself) is slowed by the binding of actin (26, repriming stroke is only significant if product release is associ- 27). The rate (AMo⅐T 3 AMc⅐T) is probably no more than 300 ated with a working stroke but for the analogues, the kinetics are sϪ1, which would lead to the pathway of dissociation before such that almost all of the events correspond to repriming and repriming being greatly favored. it is not possible to infer the working stroke associated with Physiological experiments (28) continue to indicate that the product release from trap experiments. In muscle fibers, ITP working stroke is Ϸ10 nm, considerably more than the estimates gives 35% of the tension and 15% of the shortening velocity (Ϸ5 nm) from in vitro studies. One small contribution to this provided by ATP, which in view of the majority of actin discrepancy is that Ϸ10% of the interactions of myosin with actin interactions being nonproductive and the turnover rate being result in ATP release rather than product release (6) and would slow, is consistent with the working stroke being comparable be associated with no working stroke. It is likely that the true with that of ATP. working stroke of rabbit skeletal myosin is Ϸ10% more than the In recent years there have been important developments in average displacement observed upon actin binding. relating the structures observed by x-ray crystallography and The observation that, of all of the myosin intermediate states electron microscopy to the actomyosin ATPase mechanism and investigated, M⅐D⅐P is the only one to give a working stroke leads the crossbridge cycle. First, the transition from a state with a to consideration of what constrains myosin to bind in the closed to an open switch 2 element (a feature characteristic of preforce state before undergoing the working stroke (see Fig. 9). P-loop proteins) was associated with a swing of the lever arm, There are two intuitive requirements. The first is that myosin that is, with the working stroke (90° 3 45°; reviewed in ref. 18). initially binds to actin in a preforce state; the second is that after Very recently evidence has been presented that the binding of the working stroke myosin does not dissociate from actin. The actin to Sl results in a closing of the actin binding cleft and that second requirement arises because in the postforce conforma- this is structurally coupled to the opening of switch 1 (again a tion thermal fluctuations will not be sufficient to allow rebinding

12908 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400227101 Steffen and Sleep Downloaded by guest on September 26, 2021 to the same force-producing actin monomer and another ATP (29), comfortably lower than the ATPase rate (Ϸ6sϪ1). How- will have to be hydrolyzed before force can be produced again. ever, the binding constant weakens and the rate of dissociation Meeting the first requirement is helped by the fact that the rate increases as one progresses back through the cycle to M⅐D⅐P Ϫ of Pi release is slow in the myosin ATPase scheme so that the (probably Ϸ1,000 s 1). Taylor (29) characterized the initial ⅐ ⅐ switch 2 closed (preforce) M⅐D⅐P is the dominant intermediate. AM D state formed upon M D binding to actin and the rate of Ͼ Ϫ1 However, the M⅐T state exists in open and closed forms in equal actin dissociation was 150 s . It has not been possible to prove concentrations and as the equilibrium constant of the hydrolysis that this state is on the ATPase pathway but its properties are rather similar to the AMЈ⅐D state partially characterized by step itself is close to 1, it makes it unlikely that the concentration 7 of the open form of M⅐D⅐P is negligible. In view of our measurement of ATP Pi exchange during the hydrolysis of ATP (30). observation that actin binds to the open form of M⅐T, even a A slow rate of actin dissociation goes along with tight actin small percentage of the open form of M⅐D⅐P could lead to a binding and the simplest scheme would be one in which actin was serious loss of efficiency as a result of myosin undergoing the bound tightly to a closed myosin state before the working stroke. working stroke before binding to actin. This finding suggests that This finding is in accord with the model deduced from recent some other important change in structure, associated with the crystallographic and electron microscopic observations (19–21) hydrolysis step, results in the interaction of actin with the switch in which switch 1 opens, promoting tighter actin binding before ⅐ ⅐ 2 closed form of M D P being favored. the working stroke (switch 2 opening). However, a considerable For the second requirement, the slow rate of actin dissociation fraction of the energy of ATP hydrolysis is associated with Pi from postforce states can be restated in terms of the rate of actin release (31), which means that actin binding does not really dissociation needing to be less than the overall ATPase rate. become tight until after Pi release. It is thus surprising that both Actin is tightly bound to the two states at the end of the cycle, physiological and biochemical evidence favors a working stroke AM⅐D and AM, and the rates of dissociation are Ϸ1 and 0.1 sϪ1 before the release of Pi (32, 33).

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