A Pathway of Structural Changes Produced by Monastrol Binding to Eg5*

Monastrol is a small molecule inhibitor that is specific for Eg5, a member of the kinesin 5 family of mitotic motors. Crystallographic models of Eg5 in the presence and absence of monastrol revealed that drug binding produces a variety of structural changes in the motor, including in loop L5 and the neck linker. What is not clear from static crystallographic models, however, is the sequence of structural changes produced by drug binding. Furthermore, because crystallographic structures can be influenced by the packing forces in the crystal, it also remains unclear whether these druginduced changes occur in solution, at physiologically active concentrations of monastrol or of other drugs that target this site. We have addressed these issues by using a series of spectroscopic probes to monitor the structural consequences of drug binding. Our results demonstrated that the crystallographic model of an Eg5-ADP-monastrol ternary complex is consistent with several solution-based spectroscopic probes. Furthermore, the kinetics of these spectroscopic signal changes allowed us to determine the temporal sequence of drug-induced structural transitions. These results suggested that L5 may be an element in the pathway that links the state of the nucleotide-binding site to the neck linker in kinesin motors.

The kinesins are a large superfamily of molecular motors that serve a wide variety of roles in the cell (1)(2)(3)(4). These functions are to a large extent served by different families within the kinesin superfamily. For example, although members of the kinesin 1 family transport vesicles, members of the kinesin 5 family are involved in formation and maintenance of the spindle during mitosis. The members of this latter group appear to function by generating sustained opposing forces on the overlapping microtubules that project toward each other from the two spindle poles (5)(6)(7)(8)(9)(10). Given their central role in mitosis, these motors are currently targets for the development of new generations of drugs that could be used to treat cancer (11)(12)(13) and other diseases of inappropriate mitotic activity. Monastrol is a prototypical inhibitor of Eg5 that appears to be uniquely specific for this member of the kinesin 5 family, and several laboratories have investigated its mechanism of action (14 -17). Monastrol does not inhibit ATP or microtubule binding, but it does block microtubule-stimulated ADP release. Furthermore, one study argued that monastrol enhances the reversal of the ATP hydrolysis step (14).
Insights into the structural basis for these effects of monastrol come from comparisons of crystallographic models of Eg5:ADP in the pres-ence and absence of drug (18,19). Two major effects have been reported. These include a folding of loop L5 to form the top of an induced fit, hydrophobic drug-binding cavity and a reorientation of the "switch II cluster" that is associated with docking of the neck linker (19). Loop L5 and ␣4, at the center of the switch II cluster, are separated by over 35 Å (18,19). It thus remains unclear how these conformational changes are induced by monastrol binding and how they are connected, given their wide separation.
In our previous study of Eg5 (20), we combined a series of novel spectroscopic approaches with transient kinetic measurements to develop a model of the structural changes that this molecular motor undergoes during its mechanochemical cycle. In this study, we use these and additional spectroscopic methodologies to determine the timing and the pathway of the structural changes produced in Eg5 by binding of monastrol.

EXPERIMENTAL PROCEDURES
Materials-Media components were obtained from Difco. Protease inhibitors, isopropyl D-thiogalactopyranoside, and reagents for buffers and agarose gel electrophoresis were obtained from Sigma. Ribonuclease A and deoxyribonuclease I were purchased from Roche Applied Science. Nickel nitrilotriacetic acid-agarose was obtained from Qiagen Inc. (Chatsworth, CA). Fluorescent probes were purchased from Molecular Probes. Synthesis and purification of 2Ј-deoxy-mant-ATP 3 and 2Ј-deoxy-mant-ADP from N-methylisatoic anhydride and the corresponding nucleotides were carried out as described (21). Monastrol, consisting of a mixture of the S and R enantiomers, was obtained from Calbiochem and prepared as a 5 mM stock solution in dimethyl sulfoxide. Enantiomerically pure DHPC-1R was prepared in a related study 4 using solid-phase synthesis, as described (22), and stored as a 100 mM stock solution in Me 2 SO until use. A monomeric, cysteine light mutant of Eg5, containing the first 367 residues and a single reactive cysteine in the neck linker at position 365, was generated as described in our prior study (20) and used in all of the studies described here. This construct retains the single tryptophan residue at position 127, within L5 (18). Labeling of position 365 with Oregon Green 488 maleimide was accomplished by incubating the 2ЈdmD-labeled material with a 10-fold molar excess of fluorescent probe at room temperature for 30 min. Excess fluorophore was removed by sequential equilibrium dialysis through Centricon 10 tubes and gel filtration on pre-poured Sephadex G-25 columns (PD10; Amersham Biosciences). Labeling stoichiometry was typically 0.82-0.93. Labeling of the active site of the Oregon Greenmodified construct with 2ЈdmD was accomplished by incubating Eg5 * The costs of publication of this article were defrayed in part by the payment of page with a 20 -50-fold molar excess of 2ЈdmD, incubating for 30 min at room temperature, and removing excess nucleotide by equilibrium dialysis and gel filtration as above.
Kinetic Measurements-Transient kinetic measurements were made in an Applied Photophysics SX.18 MV stopped-flow spectrometer with an instrument dead time of 1.2 ms. Complexes of Eg5 and microtubules were formed by mixing the two with a slight excess of microtubules and incubating with apyrase as described in our previous studies. Mant nucleotide to Oregon Green fluorescence resonance energy transfer was monitored by exciting the mant donor at 350 nm and monitoring the emission through a 500-nm long pass filter. Tryptophan fluorescence emission was monitored by exciting the fluorophore at 295 nm and monitoring the emission using a 335-nm long pass filter.
Transient Fluorescence Measurements-Fluorescence lifetime measurements were performed with a Fluorolog 3 spectrofluorimeter equipped with an IBH 5000 photon-counting lifetime system (Horiba Jobin Yvon, Japan). Data from donor-acceptor decays were analyzed as a static Gaussian distribution of distances, as described previously (23). Values of R 0 using Oregon Green were calculated using the quantum yield of disodium fluorescein in 0.01 M NaOH as a reference (0.91; see Ref. 24).
UV Resonance Raman Spectroscopy-Continuous wave UV resonance Raman spectra were generated using the 229 nm output of a frequency-doubled argon ion laser (1.8 milliwatts at the sample). The Raman scattered light was collected, dispersed, and detected using a previously described (25) UV Raman apparatus derived from a single spectrograph with a CCD detector. The sample-containing quartz NMR tubes were cooled (ϳ4 -10°C), spun, and vertically rastered to minimize sample heating and laser-induced degradation. The frequency scale of each spectrum was calibrated against the peaks of two reference solvents, indene and toluene, and is accurate to Ϯ1 cm Ϫ1 .

Effects of Drug Binding on Neck Linker Position and Dynamics as
Monitored by FRET and Fluorescence Anisotropy Decay-Crystallographic models of Eg5:ADP in the presence and absence of monastrol suggest that drug binding induces the neck linker to dock along the switch II complex (19), in a position analogous to what has been seen for the Kif1A-AMPPCP complex (26), conventional kinesin with bound sulfate (27), and to what has been proposed for kinesin:ATP when bound to the microtubule (28). We utilized fluorescence resonance energy transfer (FRET) to monitor the effect of monastrol binding on the neck linker to nucleotide distance distribution. We labeled our cysteine light construct at position 365 with Oregon Green 488 maleimide and at the nucleotide-binding site with 2ЈdmD, and we measured changes in FRET efficiency between these two probes in the absence and presence of monastrol and DHPC-1R, a novel Eg5 inhibitor. 4 Fig. 1 illustrates the structures of the physiologically more active enantiomers of these two drugs. DHPC-1R causes bipolar spindle collapse and displays the same kinesin family selectivity. DHPC binds to the same site as monastrol but is 8-fold more potent in cells and in vitro, with an IC 50 of ϳ1 M. 4 Furthermore, unlike monastrol, it does not have a broad absorption band in the 290 -320 nm range, which means that its presence should not interfere with measurements involving the fluorescence emission of tryptophan 127.
FRET results are summarized in Table 1. In each case, the FRET data could be fit to two distances, R 1 and R 2 , each with an associated distance distribution half-width. In the absence of drug, R 1 and R 2 had similar values, and each was associated with a fairly broad distribution halfwidth. Addition of either DHPC-1R or monastrol had no appreciable effect on R 1 , although it did increase the half-width 1.5-2-fold. By contrast, in both cases R 2 shortened by 5 Å and its half-width shortened by a factor of 3.
We also examined the effect of drug binding on the mobility of L5 by measuring the time-dependent decay of the fluorescence anisotropy of tryptophan 127. This residue is located at the apex of L5. The crystallographic model demonstrates that monastrol binding causes this loop to bend toward a hydrophobic groove formed by ␣2 and ␣3 on the surface of the motor domain, creating a hydrophobic cavity that accommodates the drug (19). Monastrol has a major absorption band centered around the tryptophan fluorescence emission, and its presence would therefore be expected to interfere with anisotropy decay measurements. We therefore utilized DHPC-1R for these studies, because it does not appreciably absorb light in the 290 -320 nm range, and as demonstrated in Table 1, this drug appears to have similar structural effects on the motor. Results are summarized in Table 2.
To determine the rotational correlation time of the entire Eg5 motor domain, we measured anisotropy decay of 2ЈdmD bound to the active site of this motor. For both the tryptophan and mant probes, two rotational correlation times could be detected. Each correlation time () is associated with an anisotropy (A). The shorter correlation time, 1 , in the range of 0.7-1.8 ns, reflects local probe motion (23), and the longer time ( 2 ) reflects movements of the domain to which the fluorophore is   attached. The value of 2 for Eg5 labeled with 2ЈdmD (49.7 ns) is similar to what had been reported previously for a similarly labeled monomeric kinesin 1 construct, and it is within the range of what would be expected for rotation of a prolate ellipsoid of the size of our Eg5 construct (23). Table 2 also demonstrates that there is significant segmental flexibility in L5 in the absence of drug, as evidenced by comparing the values of 2 for 2ЈdmD and tryptophan. Addition of DHPC-1R increases the value of 2 by nearly 60%, implying that drug binding stabilizes the conformation of L5. However, even in the presence of drug, some degree of segmental flexibility in L5 remains, as evidenced by a value of 2 that is nearly 2.5-fold shorter than that for the 2ЈdmD probe.
These conclusions are also supported by analyzing the corresponding anisotropies, A 1 and A 2 . The values of these parameters are related to the semi-cone angle of the fluorophore, which represents the angular distribution of possible orientations that the fluorophore can assume. The relationship between the anisotropies and the semi-cone angle, , is described by Equation 1 (29), Solving for leads to several conclusions (Table 2). First, there appears to be a moderate amount of rotational freedom for the mant fluorophore. This is consistent with the surface exposure of the 2Ј-ribose hydroxyl group evident in crystallographic models of Eg5 (18,19). It is also consistent with our prior quenching studies, which revealed appreciable solvent exposure for the mant fluorophore when bound to kinesin 1 (30). Second, the rotational freedom of tryptophan 127 in the absence of drug is appreciably larger than that for the mant fluorophore, a finding that is consistent with the corresponding differences in rotational correlation times. Finally, binding of DHPC-1R reduces for tryptophan 127 to a value that approximates that for the mant probe (Table 2). This supports our conclusion from the rotational correlation time measurements that drug binding to L5 reduces its mobility. Effect of Monastrol on the Conformation of L5, Monitored by UV Resonance Raman Spectroscopy-Monastrol binding has been reported to produce an ϳ7-Å movement of L5, leading to its folding over to produce a hydrophobic binding pocket for drug. This is illustrated in Fig. 2, which superimposes the drug-free (red) and drugbound (yellow) conformations, derived from crystallographic models (18,19). To determine whether the same conformational changes occur under physiologic conditions, in solution, we examined the effects of monastrol binding on the Raman spectrum of the single tryptophan residue of Eg5. As illustrated in Fig. 2, tryptophan 127 lies at the apex of L5, and drug binding induces it to move into a hydrophobic environment. Raman spectra in the presence and absence of drug are depicted in Fig. 3. An increase in the intensity of the W3 band, located at 1550 cm Ϫ1 , is seen with addition of monastrol ( Fig.  3, dashed line) and indicates that drug binding causes the indole ring to move to a more hydrophobic environment (25,31). By contrast, the frequency of the W3 band is unaffected by monastrol binding. This indicates that the dihedral angle for tryptophan, which is the torsional angle between the pyridine ring and the aliphatic linker to the protein backbone, is the same for the drug-free and drug-bound states (31). These conclusions are consistent with the crystallographic models in the presence and absence of drug (Fig. 2).
Additional changes induced by monastrol binding are also seen in resonances that reflect changes in the hydrogen bonding status of phenolic hydroxyls. These are best seen in the tyrosine Y8 band at 1614 cm Ϫ1 . However, a comparison of the crystal structures in the presence and absence of monastrol reveals that multiple tyrosine residues change their degree of phenolic hydroxyl hydrogen bonding in response to drug  binding (25,31), and this limits our ability to provide a structural interpretation to these spectroscopic changes. Although monastrol has a phenolic side chain, its Raman spectrum has only a low intensity peak in this part of the spectrum that does not appreciably contribute to the Raman intensities described above (data not shown).
Kinetics of Monastrol Binding to Eg5:ADP-The strong absorption band of monastrol in the 290 -320 nm range allows us to monitor the kinetics of monastrol binding to Eg5:ADP, through its quenching of the Eg5 tryptophan at position 127. This spectroscopic strategy was used in a prior study that demonstrated that mixing Eg5:ADP with monastrol quenched the tryptophan fluorescence emission in a single phase (32). However, commercially available monastrol is a racemic mixture of equimolar concentrations of S and R enantiomers, which have different affinities for an Eg5 intermediate (17). We therefore wished to see if we could distinguish the binding of these two enantiomers by re-examining the kinetics of the quenching of tryptophan 127. Results are depicted in Fig. 4A. The fluorescence transient resulting from mixing Eg5:ADP with monastrol consists of two phases. The rate constants of both the fast and slow phases depend hyperbolically on monastrol concentration (Fig. 4A,  inset). This dependence defines maximum rate constants for binding of 536 Ϯ 139 and 39 Ϯ 3 s Ϫ1 and for dissociation of 82 Ϯ 25 s Ϫ1 and 3 Ϯ 1 s Ϫ1 . Furthermore, the apparent monastrol affinities, determined from the hyperbolic fitting, are 96 Ϯ 37 M for the fast phase and 12 Ϯ 3 M for the slow. Finally, the relative amplitude of the faster phase increases hyperbolically with drug concentration, extrapolating to 0.97 Ϯ 0.05 (Fig. 4B).
These results are consistent with the mechanism depicted in Scheme 1, where the boldface Eg represents Eg5:ADP, and the species in parentheses represent a collisional complex of Eg5-ADP with S monastrol (M S ) or R monastrol (M R ). For both enantiomers, formation of a collisional complex, characterized by dissociation constants K S and K R , is followed by a conformational change, which is approximately 10 times faster for one of the enantiomers. Given the lower affinity of R monastrol for Eg5 in solution (17), we propose that the faster phase in the transient depicted in Fig. 4A reflects initial binding of R monastrol. This scheme would predict that at high monastrol concentrations ([monastrol] Ͼ Ͼ K R ), the relative amplitude of the faster phase (A r ) would approach the value defined by Equation 2, Given the extrapolated rate constants in Fig. 4A, we predict A R to be 0.93, which compares to the extrapolated value in Fig. 4B of 0.97 Ϯ 0.05. For both enantiomers, formation of the initial collisional complex is followed by a change in conformation of L5, which brings the tryptophan in closer proximity to the drug.
Kinetics of Monastrol-induced Reorientation of the Neck Linker-Our results using FRET from 2ЈdmD in the catalytic site to Oregon Green 488 at position 365 in the neck linker (Table 1) would lead us to predict that monastrol binding should produce an increase in FRET efficiency and therefore in the sensitized emission of the Oregon Green acceptor, with drug binding. This is confirmed in Fig. 5A. Mixing of labeled Eg5: ADP with monastrol produces an increase in Oregon Green fluorescence due to energy transfer from the mant donor (Fig. 5A, Experiment). Repeating this experiment in the absence of the mant fluorophore, e.g. with Eg5:ADP instead of Eg5:2ЈdmD, produces no fluorescence increase (Fig. 5A, Control). Both the rate constant for this fluorescence increase (Fig. 5B) and the amplitude (Fig. 5C) depend hyperbolically on monastrol concentration. This dependence defines a maximum rate of 0.68 Ϯ 0.07 s Ϫ1 and apparent dissociation constants of 63 Ϯ 22 and 41 Ϯ 6 M for Fig. 3, B and C, respectively.

DISCUSSION
Although Eg5 shares many features with kinesin 1, these two motors differ structurally in several respects. One of the most striking of these is in the length of L5, which is substantially longer in Eg5 (18). This difference in particular may explain why a variety of different small molecule inhibitors, including monastrol, S-trityl L-cysteine, and DHPC, can specifically bind to and inhibit Eg5, because each appears to interact at least in part with L5 (19,33). 4 Crystallographic studies have demonstrated that monastrol binding induces L5 to fold over and form a drug-binding pocket. This also occurs with binding of S-trityl L-cysteine (33), and it is likely to be a common mechanism by which other small molecule inhibitors, such as DHPC, specifically block Eg5 as well. 4 In addition to its effects on L5, monastrol binding also induces widespread changes in the structure of other important domains. These include switch I, the switch II cluster, and the neck linker (19). Furthermore, monastrol also changes the equilibrium constant for ATP hydrolysis in the active site (14). These effects suggest that the conformation of L5 may control the state of both the nucleotide and microtubule-binding sites along with the orientation of the neck linker. In this study, we have used a variety of spectroscopic approaches to verify if these conclusions, derived from crystallography, are consistent with the behavior of the motor in solution. In addition, we have applied these approaches to kinetic studies to determine the timing of these structural changes.
Although the data for FRET between 2ЈdmD in the catalytic site and Oregon Green in the neck linker (Table 1) could be fit to a single dis-tance, the value we obtained in the absence of drug (ϳ61 Å) was too large to be realistic. If we fit instead to two discrete distances, R 1 and R 2 (Table 1), we obtained better fitting with distances that were more realistic. Fitting to two distances is also consistent with our previous study (20), where we found kinetic evidence to suggest that in the presence of ADP, the Eg5 neck linker is in equilibrium between two orientations. As Table 1 also shows, both R 1 and R 2 are characterized by large halfwidths. Taken together, these results suggest that in the absence of drug, the neck linker alternates between two orientations, each of which is characterized by an appreciable amount of disorder.
Drug binding has no appreciable effect on R 1 , although the half-width did increase. By contrast, R 2 was found to shorten to a value similar to that seen in the crystallographic model of Eg5:ADP:monastrol (19), a structure in which the neck linker is docked along the motor surface. Furthermore, drug binding also markedly shortens the half-width of the R 2 distance distribution, suggesting that it also reduces the disorder or mobility of the neck linker. We interpret these results to mean that drug binding favors formation of a stable docked orientation of the neck linker, consistent with the crystallographic model (19). We have no direct measure of what the other undocked orientation, characterized by distance R 1 , looks like. We do note that the value of R 1 , at 55 Å, is considerably longer than that reported in the crystallographic model in the absence of drug (38.4 Å; see Ref. 18). We explain this apparent discrepancy as follows. In the absence of drug, the neck linker functions as a rigid lever that rotates on a pivot located at or near its junction with ␣6. We propose this for two reasons. First, in our previous study (20), we found that although the neck linker of a rigor Eg5-microtubule complex behaved as if it were an extended rod, similar measurements with kinesin 1 suggested that its neck linker was collapsed and disordered, consistent with previous studies (28). Second, the unexpectedly large value of R 1 implies that the Eg5 neck linker can at least briefly assume an extended conformation. We propose that in solution, rotation around this pivot is modulated by two relatively shallow energy minima, one favoring an orientation with the neck linker close to the motor surface, characterized by R 2 , and one favoring a more extended orientation in which the neck linker projects away from the motor surface, characterized by R 1 . Drug binding would favor a docked orientation, leading to reduction in R 2 and in a marked reduction in its half-width. The conformational changes in the motor domain induced by drug, which stabilize the docked orientation (19), would also be expected to further reduce the stability of the extended orientation. This effect would increase the disorder of the extended orientation and would produce an increase in the half-width of R 1 (Table 1). Finally, we propose that in the crystal of Eg5:ADP (18), packing forces provide additional stability to the neck linker, shortening its distance to the catalytic site, reducing its angular disorder, and explaining the difference between our value of R 1 and the crystallographic distance.
Our transient kinetic results are also consistent with this model, because they would predict that the average distance between the Oregon Green and 2ЈdmD probes should shorten with drug binding. As Fig.  5A demonstrates, mixing Oregon Green-labeled Eg5:2ЈdmD with monastrol increases the Oregon Green-sensitized emission, as predicted by our model.
Our finding that the quenching of tryptophan 127 by monastrol occurs in two phases is consistent with the fact that our monastrol preparations are racemic mixtures of R and S enantiomers. Our kinetic results (Fig. 4) suggest that the maximum rate constants for both complex formation and those for drug dissociation are at least 10 -20-fold faster with the R enantiomer. Furthermore, the apparent affinity for monastrol, determined from the kinetics of neck linker docking (Fig. 5, A-C), is intermediate between those we have assigned to the R and S enantiomers. This implies that both enantiomers are capable of docking the neck linker. In the absence of monastrol or DHPC, L5 is quite mobile, as demonstrated both by the high temperature factor in crystallographic models (19) as well as the rotational correlation time and semi-cone angle of tryptophan 127 (Table 2). However, even in the presence of DHPC, the rotational correlation time of tryptophan 127 remains 2.5-fold shorter than that for the entire molecule. Furthermore, its semi-cone angle, although reduced in the presence of drug, approximates that for the mant probe, which we have shown previously is solvent-exposed and flexible (30). This suggests that L5 retains a significant amount of segmental flexibility in all states, a degree of flexibility that might be necessary to accommodate the binding of different enantiomers of monastrol. These conclusions are supported as well by the resonance Raman spectra of Eg5 in the presence and absence of monastrol. Spectroscopic differences induced by addition of monastrol are consistent with the crystallographic model and support the conclusion that drug binding causes tryptophan 127, at the apex of L5, to move into a hydrophobic environment (Fig. 3).
In a separate study, DeBonis et al. (Figs. 7-9 in Ref. 17) mixed mant-ADP-labeled Eg5 in the stopped flow with ATP and monastrol and also observed a biphasic fluorescence decay, reminiscent of our results in Fig.  4A. They attributed their findings to a three-step monastrol binding reaction. However, in their experiments, they monitored the fluorescence emission of the mant fluorophore by energy transfer from both Eg5 tryptophan and tyrosine residues. This makes it very difficult to interpret their kinetics in terms of specific structural transitions. Furthermore, they monitored the effect of monastrol binding by its interference with energy transfer from these tyrosine and tryptophan residues to the mant fluorophore. Again, this experimental design makes it very difficult to ascribe changes in sensitized acceptor emission to specific, monastrol-induced structural changes. Finally, these authors did not consider the possibility of separate contributions from the S and R enantiomers of monastrol in their experiment, even though their experiment was performed with an enantiomeric mixture. By contrast, our data, depicted Fig. 4, were generated by monitoring monastrol-induced quenching of the emission from the tryptophan 127 donor alone. It therefore provides results that are structurally interpretable and complementary to those from UV resonance Raman spectroscopy (Fig. 3). Furthermore, our results are entirely consistent with the effects expected from using an equimolar mixture of S and R enantiomers of monastrol.
The rate constant for neck linker docking, as measured by our mant 3 Oregon Green FRET pair, is considerably smaller than that for monastrolinduced L5 folding. We can therefore use our kinetic results to propose the temporal sequence of conformational changes that follow the binding of monastrol. Comparisons of the two crystallographic models suggest that monastrol binding induces folding of L5, translation of ␣3, reorientation of switch I and II, and neck linker docking. Given the relative values of the rate constants for the transitions depicted in Figs. 4 and 5, we propose that folding of L5 occurs first and is only later followed by the changes in switch II that favor neck linker docking.
Kinesin 1 and Eg5 differ structurally in several respects, most significantly in the length of L5 (18). In our prior study, we demonstrated that the rate of neck linker docking by Eg5 is nearly 20-fold slower than that for kinesin 1 (20). If the conformation of L5 controls the state of the neck linker, then varying L5 length and flexibility may affect how quickly it can change conformation and, consequently, how quickly the neck linker can dock. This in turn may explain how the kinesin superfamily modulates the speed of force generation in different motors that subserve different physiologic functions. This concept is illustrated in the case of myosin II, where variations in the size and flexibility of loop 1 directly alter the kinetics of nucleotide binding, release, and presumably, force generation (34). Our results thus speak to the importance of L5 in modulating kinesin behavior and suggest that further studies, which dissect the nature of interactions between this loop and other important structural domains in Eg5, will yield valuable insights into how this motor functions.