Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2285-2289, March 1996 Biochemistry

The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head (heavy meromyosin/limited tryptic proteolysis/velocity of movement)

ANDREY A. BOBKOV*, ELENA A. BOBKOVAt, SHWU-HWA LINt, AND EMIL REISLER* *Department of Chemistry and Biochemistry and the Molecular Biology Institute, and tDepartment of Physiology, School of Medicine, Center for Health Sciences, University of California, Los Angeles, CA 90095; and tCenter for Ulcer Research and Education, Department of Medicine, University of California at Los Angeles School of Medicine and Wadsworth Division, Department of Veterans Affairs Medical Center, West Los Angeles, CA 90073 Communicated by James A. Spudich, Stanford University, Stanford, CA, December 4, 1995 (received for review August 17, 1995)

ABSTRACT A characteristic feature of all myosins is the A characteristic feature of all myosins is the presence of two presence of two sequences which despite considerable varia- sequences which can be aligned with loops 1 (residues 204- tions in length and composition can be aligned with loops 1 216) and 2 (residues 627-646) in the chicken myosin-head (residues 204-216) and 2 (residues 627-646) in the chicken heavy chain sequence. The first loop is located in the vicinity myosin-head heavy chain sequence. Recently, an intriguing of the active site on the myosin subfragment 1 (S1) (2), while hypothesis has been put forth suggesting that diverse perfor- loop 2 is part of the actin-binding interface (2, 3-6). Strikingly, mances of myosin motors are achieved through variations in the length and sequence of these loops vary considerably the sequences of loops 1 and 2 [Spudich, J. (1994) Nature among different myosins (7). In general, variations in protein (London) 372, 515-518]. Here, we report on the study of the sequences are considered to be clustered in functionally un- effects of tryptic digestion of these loops on the motor and important regions. Recently, an intriguing hypothesis has been enzymatic functions of myosin. Tryptic digestions of myosin, put forth suggesting that the performance of different myosin which produced heavy meromyosin (HMM) with different motors is achieved through variations in the sequences of loops percentages of molecules cleaved at both loop 1 and loop 2, 1 and 2 (8). Strong support for this hypothesis comes from the resulted in the consistent decrease in the sliding velocity of considerable evidence on the involvement of surface loop 2 in actin filaments over HMM in the in vitro motility assays, did the binding of actin to myosin (3-6). Strikingly, experiments not affect the Vmax, and increased the Km values for actin- with chimeric Dictyostelium myosins, in which loop 2 was activated ATPase of HMM. Selective cleavage of loop 2 on substituted by loop sequences from other myosins, showed a HMM decreased its affinity for actin but did not change the good correlation between the actin-activated ATPase activities sliding velocity of actin in the in vitro motility assays. The of these chimeras (at single actin concentration) and the cleavage of loop 1 on HMM decreased the mean sliding activities of parent myosins (9). At the same time, the in vitro velocity ofactin in such assays by almost 50% but did not alter motilities of actin filaments determined for the chimeric its affinity for HMM. To test for a possible kinetic determi- myosins did not show any correlation with the motor velocity nant of the change in motility, 1-N6-ethenoadenosine diphos- of loop 2 parent myosins. These results are consistent with the phate (eADP) release from cleaved and uncleaved myosin proposed involvement of loop 2 in actomyosin ATPase (4, 6, subfragment 1 (S1) was examined. Tryptic digestion of loop 1 8) but also focus attention on the lack of any direct correlation slightly accelerated the release of eADP from S1 but did not between changes in the enzymatic (solution actomyosin AT- affect the rate of EADP release from acto-Sl complex. Overall, Pase) and motor (in vitro actin motilities) velocities of myosin. the results of this work support the hypothesis that loop 1 can The lack of such correlation was shown also by other authors modulate the motor function of myosin and suggest that such (10, 11) and suggests that actin-activated ATPase and in vitro iiodulation involves a mechanism other than regulation of motility assays may have different rate-limiting steps (10). ADP release from myosin. Very little is known about the possible functional role of surface loop 1. It was shown that the cleavage of loop 1, in Cyclic interactions of myosin heads with actin filaments cou- contrast to the cleavage of loop 2, does not affect the actin- pled to ATP hydrolysis are the molecular basis of muscle activated ATPase activity of S1 (12, 13). According to the contraction. It is believed that the chemical energy from ATP myosin-loop hypothesis, this and other results can be accom- hydrolysis is transduced into mechanical force and movement modated by assigning a role in actomyosin ATPase to loop 2 through conformational changes in the myosin head. The and a motor-velocity-modulating function to loop 1 (8). actomyosin-ATP hydrolysis cycle can be simplified as de- It is known that trypsin can cleave both loop 1 and loop 2 of scribed by Scheme 1 (1). the myosin head (14). In this study, we examine the effects of such cleavages on the enzymatic and motile functions of 1 2 3 4 myosin to clarify the possible role of these loops in the AM + ATP AM*ATP AM*ADP-Pi <-* AM-ADP + Pi AM + ADP molecular mechanism of muscle contraction. $ 2' $ MATP MADP*Pi, MATERIALS AND METHODS Scheme 1 Proteins. Myosin and actin from back and leg muscles of where AM is actomyosin and M is myosin. It is assumed that rabbits were prepared according to Godfrey and Harrington force generation occurs during the transition of the actomyosin (15) and Spudich and Watt (16), respectively. S1 was prepared complex from the weekly bound state (AM-ADP.Pi) to the strongly bound states (AM-ADP, AM). Abbreviations: Si, myosin subfragment 1; S2, myosin subfragment 2; HMM, heavy meromyosin; cLl-HMM, cL2-HMM, cL1,2-HMM, HMM with loop 1, loop 2 or both loops cleaved, respectively; cLi-Sl, The publication costs of this article were defrayed in part by page charge subfragment 1 with cleaved loop 1; sADP, 1-N6-ethenoadenosine payment. This article must therefore be hereby marked "advertisement" in diphosphate; F-actin, filamentous actin; LC1, -2, and -3, myosin light accordance with 18 U.S.C. §1734 solely to indicate this fact. chain 1, 2, and 3, respectively. 2285 Downloaded by guest on September 25, 2021 2286 Biochemistry: Bobkov et al. Proc. Natl. Acad. Sci. USA 93 (1996) by digestion of myosin filaments with a-chymotrypsin (17). the same reaction. Fig. 1 shows SDS/gel electrophoretic Chymotryptic and tryptic heavy meromyosin (HMM) were patterns of HMM species produced after different digestion prepared according to Margossian and Lowey (18). In the times. The major products in the first minutes of digestion (Fig. latter case, digestion times varied between 1 and 60 min to 1, lanes b and c) are the 75-kDa and 67-kDa fragments, which produce HMM with different percentages of molecules result from HMM cleavage at loop 2. During further digestion cleaved at loop 1 and loop 2. The concentrations of myosin and the 67-kDa fragment degrades to 63 kDa and finally into the trypsin in these digestions were 10 mg/ml and 0.05 mg/ml, 60-kDa fragment. Previous study showed that myosin subfrag- respectively. Insoluble myosin fragments (light meromyosin) ment 2 (S2) was progressively degraded from its C terminus by were identified by electrophoresis of supernatant and pelleted trypsin from a fragment of 53 kDa to one of between 35 and fractions of tryptic digestion products that had been dialyzed 40 kDa (26). Thus, degradation of the 67-kDa product corre- into 40 mM NaCl/5 mM Pi, pH 6.5. sponds to the degradation of the S2 part of this fragment. The Tryptic Proteolysis of Chymotryptic HMM and Si. In all 75-kDa fragment is split during further digestion into the digestions, the reaction mixture included 20 mM KCl, 20 mM 50-kDa and 27-kDa fragments, which are produced by the Tris-HCl (pH 7.5) or 20 mM Pipes (pH 7.0) a final trypsin cleavage of loop 1. As shown in Fig. 1, loop 2 was almost concentration of 0.05 mg/ml and 3 mg of Si or HMM per ml. completely split by trypsin after 4 min of digestion (lane d), Digestion was carried out for 8 min at 20°C. ATP-protected while loop 1 was not completely split until -20 min (lane f). digestion of HMM was carried out in the presence of 6 mM Thus, in agreement with a previous study (13), trypsin cleaves MgATP and 0.5 M KCl (19); actin-protected digestion was loop 2 faster than loop 1 on HMM. carried out in the presence of a 2-fold molar excess of Sliding velocities of actin filaments in the in vitro motility filamentous actin (F-actin) over S1 (13). assays were measured for all HMM species shown in Fig. 1. Fig. Gel Electrophoresis. SDS/10% PAGE was carried out accord- sliding velocities for ing to Laemmli (20). Molecular masses of protein fragments were 2 shows the distribution of filament determined by comparing their electrophoretic mobilities to selected species of the tryptically cleaved HMM (Fig. 2, b, d those of marker proteins. and f). Clearly, filament sliding velocities decrease with in- Actin-Activated ATPase Activities of HMM. Actin-activated creasing tryptic digestion of myosin. Importantly, HMM af- ATPase activities of HMM were determined under steady- finity for actin also weakens during this digestion. All HMMs state conditions by colorimetric assays in a solution containing obtained by digestions longer than 1 min required methylcel- 10 mM imidazole (pH 7.0), 10 mM KCl, 3 mM MgCl2, 3 mM lulose to avoid diffusion of actin filaments from the HMM- ATP, 0.1 ,uM HMM, and between 10 and 100 ,tM F-actin at coated surface in the presence of ATP. Table 1 presents the 25°C. Since such measurements are difficult at actin concen- mean velocities of actin filaments for HMM species which were tration >100 ,xM, Km values >100 ,uM are considered to be produced by tryptic digestion of myosin over different periods only estimates of this parameter. of time. Notably, the mean actin velocities decrease during In Vitro Motility Assays. In vitro motility assays were per- tryptic digestion of myosin but not to zero. After a 60-min formed at 25°C, as described (21), with freshly prepared digestion of myosin, the sliding of actin filaments could not be proteins (prepared 1 or 2 days previously). Each HMM measured since they diffused away from the HMM-coated derivative was centrifuged with F-actin in the presence of surface even in the presence of methylcellulose. MgATP to remove inactive HMM molecules. Movement of Measurements of actin-activated ATPase activities for se- actin was initiated with assay buffer (25 mM KCl/1 mM lected tryptic HMM species (Table 1) showed that the tryptic MgCl2/10 mM dithiothreitol/25 mM Mops, pH 7.4) contain- digestion had little effect on Vmax but significantly weakened ing 1 mM ATP and oxygen-scavenging system in the presence the affinity of HMM to actin (Km increased up to -200 ,uM). or absence of 0.7% methylcellulose. Quantification of sliding As shown in Fig. 1, loop 2 (the actin-binding loop) was velocities was achieved with an Expertvision system (Motion completely split after 4 min of digestion (lane d). Yet, the Km Analysis, Santa Rosa, CA). for actin-activated ATPase continued to increase with addi- Stopped-Flow Measurements. The stopped-flow measure- tional digestion. This result can be explained by the previously ments were carried out in a Hi-Tech PQ/SF-53 sample- observed presence of two tryptic sites in loop 2 (the 50-60 kDa handling unit. The Hi-Tech mixer is integrated into an On- Line Instrument Systems spectrophotometer. The instrument C-HMM ' 2' 4' 10' 20' 60' is interfaced to an AST Premium 286 computer. All experi- ments were performed according to the method of Rosenfeld I]-Ic and Taylor (22) at 15°C in a solvent consisting of 3 mM MgCl2, 75 kDa 10 mM KCI, 10 mM Pipes (pH 7.0), and 100 mM acrylamide .-67 kDa by rapid mixing of solutions from two syringes. One syringe |='-: 63 kDa contained 10 ,tM S1 and 20 ,uM 1-N6-ethenoadenosine diphos- 6() kDa phate (sADP); the second syringe contained either 200 ,uM \50 kDa ADP or F-actin and 200 ,uM ADP. F-actin concentration in the second syringe was 10, 20, 40, or 80 ,uM. The kinetic curves were analyzed as described (23). Each observed rate constant is an average of between 10 and 15 measurements. S1 was used kDa instead of HMM as a simpler and more convenient material for 27 LC'I_ solution experiments; these two myosin fragments share the I.C''\ same kinetic mechanism of ATPase cycle (24, 25). eADP was 1,C3 chosen as an ADP analog for these experiments. It was shown before that dissociation of sADP from acto-Sl complex could a b c d e f g be described by kinetic scheme similar to the one describing the dissociation of ADP, and the dissociation of ADP is much FIG. 1. Representative SDS/PAGE patterns of HMM prepara- tions from myosin. Lane a, HMM prepared by chymotryptic digestion more difficult to measure (22). of myosin. Lanes b-g show HMM obtained after tryptic digestions of myosin for 1 (b), 2 (c), 4 (d), 10 (e), 20 (f) or 60 (g) min, respectively. RESULTS Digestion times are indicated above each lane. The molecular masses of HMM fragments are given on the side of the gel; the symbols HC, Tryptically Cleaved HMM. We used tryptic digestions of LC 1-3, and C-HMM correspond to HMM heavy chain, myosin light myosin to produce HMM and to cleave loop 1 and loop 2 in chains 1-3, and chymotryptic HMM, respectively. Downloaded by guest on September 25, 2021 Biochemistry: Bobkov et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2287 kDa did not slow the HMM motor function. The degradation 20 -b of LC1 by trypsin occurs from the N terminus and results in the LC3-like product. Because the myosin isoform containing LC3 was shown to move actin filaments in the in vitro motility assay -50% faster then myosin-LC1 (30), the degradation of LC1 to o-) nl111 1_III the LC3-like product could not account for the decreased C 01-d movement velocity in our assays. Finally, as shown in Fig. 1, E d0 LC2 cleavage was completed within the first minute of diges- 40 tion and, thus, showed no correlation with the continued 0 decrease in the sliding velocity of actin (Table 1). This does not completely exclude the possibility that a further, slower diges- )Z 20 HHHn tion of LC2 might affect the movement velocity. These con- siderations suggest that the decreased movement velocity of tryptically cleaved HMM could be related to the cleavage in loop 1, to the weakening of HMM binding to actin (via the ~~H[][]FHH~ I' I cleavage at the second tryptic site in loop 2) or, with a lower 1 23 4 5678 probability, to the digestion of LC2. To examine these possi- Velocity (gm/sec) bilities, we produced HMM selectively cleaved at either loop 1 or loop 2. FIG. 2. Distribution of velocities of actin filaments sliding in the in Tryptic Digestions of Protected HMM: Loop 1- and Loop vitro motility assay over HMM produced by tryptic digestion of myosin 2-Cleaved HMM. Loop 1, which is close to the ATP-binding for 1 (b), 4 (d) and 20 (f) min. SDS/PAGE patterns of these samples site on the myosin head, can be protected from tryptic are shown under the same symbols in Fig. 1. digestion by high ionic-strength conditions and the presence of is in the of junction in HMM) (27). Digestion at the first site (between ATP (19). Loop 2, which actin-binding site myosin in myosin heavy chain se- head, is protected from tryptic digestion by F-actin (13). We Lys-635 and Lys-636 the chicken to cleaved at quence) generates the 22-kDa and 50-kDa fragments of Si (13) used these conditions produce HMM selectively and does not affect its actin-activated ATPase activity (13, 28). either loop 1 (cLl-HMM) or loop 2 (cL2-HMM). The prop- During further digestion, at the second tryptic site (between erties of such HMM species were compared with those of Lys-639 and Lys-640), the 22-kDa fragment degrades into a intact HMM and HMM cleaved at both loops (cL1,2-HMM). 20-kDa fragment. The consequent loss of a four-residue SDS/polyacrylamide gel electrophoretic patterns corre- peptide (636-639) from loop 2 significantly decreases SI sponding to HMM, cL2-HMM, cLl-HMM, and cLl,2-HMM affinity for actin (13, 28). In the case of HMM the 67- to are presented in Fig. 3. The protein bands which identify these 60-kDa fragments are composed of the C-terminal 20- to materials are the 75- (50 + 27) and 67-kDa fragments resulting 22-kDa fragment of S1 and the 40- to 50-kDa fragment S2. The from loop 2 cleavage in cL2-HMM, the 115- (50 + 60) and C-terminal degradation of the S2 part of HMM makes it 27-kDa fragments due to loop 1 cleavage in cLl-HMM, and the difficult to detect on SDS/polyacrylamide gels the second 60-, 50-, and 27-kDa bands due to cleavages at both loop 1 and cleavage in loop 2. Such a cleavage is probably included in the loop 2 in cLl,2-HMM. degradation of the 67-kDa fragment into a 60-kDa fragment, Distributions of filament sliding velocities in the in vitro resulting in the weakening of HMM affinity for actin. motility assays for HMM, cL2-HMM, cLl-HMM, and cLl,2- The above results left open the question about the mecha- HMM are shown in Fig. 4. Clearly, the velocity of cLl-HMM nism for the inhibition of myosin motor velocity by tryptic is similar to that of cLl,2-HMM, while the velocity of cL2- digestion. As shown in Fig. 1, a degradation of myosin light HMM is close to that of HMM. All three HMM derivatives chains 1 and 2 (LC1, LC2) and the S2 part of HMM occurs were obtained by tryptic digestions of equal duration and during tryptic digestion of myosin in addition to the cleavages showed the same degree of LC2 degradation (Fig. 3). This in loops 1 and 2. In principal, all these events could decrease excludes LC2 degradation as a possible cause for the velocity the movement velocity in the in vitro motility assays. However, differences between cLl-HMM and cL2-HMM and suggests it is unlikely that the degradation of S2 or LC1 and LC2 affect that the cleavage at loop 1 is responsible for a decrease in the the motor function in our experiments. It was shown previously velocities of actin movement. Despite the strong effect on (29) that the degradation of S2 by trypsin from 55 kDa to 35 115 kDai Table 1. Mean velocities of actin in the in vitro motility assays and / 75 kDai 4b~ actin-activated ATPase for tryptic preparations of HMM ...... 7/ 67 kDa from myosin Myosin Actin-activated ATPase * 5( kDa digestion Motility. ,m/s 11(' _ time, min Mean velocity SD SEM Kin, /LM Vmax, S- 1 3.55 1.27 0.10 41 + 4 11 + 1.1 2 3.45 1.04 0.06 4 3.01 0.99 0.05 27 kDa 10 2.29 1.27 0.09 130 9.4 + 0.9 20 2.46 0.90 0.09 ...... , 60 - 200 9.0 0.9 I-C3_...... a b e (I All tryptic HMMs were prepared by tryptic digestion of myosin over different times to produce HMM samples with different percentages FIG. 3. Representative SDS/PAGE patterns of selectively cleaved of cleavage at loops 1 and 2. The velocities of more than 100 actin HMM. Lane a, uncleaved HMM; lane b, cL2-HMM; lane c, cLl- filaments were analyzed for each HMM preparation. Each experiment HMM; and lane d, cLl,2-HMM. Selectively cleaved HMM species was repeated three times; the absolute values of mean velocities varied were prepared as described in Materials and Methods. HC, HMM heavy somewhat depending on the age of the proteins. chain. Downloaded by guest on September 25, 2021 2288 Biochemistry: Bobkov et al. Proc. Natl. Acad. Sci. USA 93 (1996)

1 2 3 4 5 6 otide-binding site on Si, could affect the motor function of myosin by altering its interactions with ADP (8). To test this 1oo a suggestion, apparent rate constants for the release of eADP, a fluorescent ADP analog, from Si (k0ff, si) and acto-Sl 50 complex (koff, A-Si) for uncleaved Si and Si cleaved at loop 1 (cLi-S1) were measured at 15°C by using stopped-flow meth- 0. -...... ods. The value of k.ff, si for uncleaved S1 (0.98 ± 0.01 s-1) was b- similar to that previously reported (32), the eADP release from 50 cLi-Sl was slightly faster (koff si = 1.65 + 0.01 s-1). However, this difference can not account for different motor velocities E of cleaved and uncleaved HMM. m 0 eADP release from acto-Sl complexes was measured at 02 c several actin concentrations. At the highest final concentration -0 25 of 40 ,tM actin, which is close to the saturation of unfraction- z ated Si (Al and A2 isoforms of Si) by actin (22), koff, A-SI values for Si and cLl-Si were 78.4 ± 2.5 s-1 and 78.5 ± 5.1 0 s-1, respectively. Under these conditions, the previously re- d -- ported rate constants of -ADP release from S1 were between 20 10 and 20 s-1 at 5°C and 400 and 500 s-1 at 20°C (1, 33). Our results fall within the range of previous observations. Impor- tantly, the cleavage of loop 1 did not affect the rates of sADP 1 2 3 4 5 6 release from S1 at any final concentration of actin used in this work (up to 40 j,M). Since sADP and ADP have a similar Velocity (pim/sec) kinetic mechanism of dissociation from acto-Sl complex (23), FIG. 4. Distribution of velocities of actin filaments sliding in the in it can be assumed that the rate of ADP release from acto-Si vitro motility assay over uncleaved HMM (lane a), cL2-HMM (lane b), is not affected by the cleavage at loop 1. cLl-HMM (lane c), and cLl,2-HMM (lane d). These results reveal that the changes in the motor functions of S1 caused by the tryptic cleavage at loop 1 can not be mean actin velocity, the cleavage of loop 1 does not affect the attributed to changes in the rate of ADP release from the Vma, and Km for actin-activated ATPase of HMM (Table 2). acto-Sl complex. Unchanged Km for cLl-HMM eliminates the possibility that the slower movement is due to changes in the affinity of HMM DISCUSSION to actin. Vm. , values for the four HMM species in Table 2 were virtually the same, showing again the lack of a direct correla- The main result of this study is the observation that tryptic tion between the Vm, of actin-activated ATPase and the motor cleavage of loop 1 on myosin head inhibits the motor function velocity in the in vitro motility assay. The digestion of loop 2 of myosin without any significant changes in the Km and Vmax caused an increase in Km for actin-activated ATPase (Table 2), values of actomyosin ATPase, while the cleavage of loop 2 reflecting the weakening of HMM affinity for actin. This was increases the Km value with little if any impact on the motor manifested also in the in vitro motility assays in which actin velocity. These findings support the recent hypothesis (8) on filaments diffused away from the cL1,2-HMM-coated surface the functional roles of the two myosin head loops and the and, to a smaller extent, from the cL2-HMM-coated surface. implications of their sequence divergency to the enzymatic and In the latter case (cL2-HMM), the damage to loop 2 was mechanical performance of different myosins. smaller than that when using cLl,2-HMM (see Km values in Prior in vitro motility studies on tryptically cleaved myosin Table 2). Methylcellulose was required to support normal actin used S1 as the motor protein (34, 35) and did not focus on movement over these two cleaved HMM preparations but had cLl-Sl (35). A weakness of the S1 system is that its somewhat no effect on actin movement over HMM or cLl-HMM. unsatisfactory and erratic motor function may be related to its EADP Interaction with Si and Acto-Sl Complex. The adsorption to the coverslip surface via functionally significant observed involvement of loop 1 in determining the mechanical sites. The cleavage of S1 by trypsin may further compound this velocity of the myosin motor but not its catalytic velocity (Vmax) problem. Certainly, the impact of such digestions on S1 in the presence of actin is consistent with the possibility that adsorption to surface is unknown. Thus, a comparison of S1 the two processes are rate-limited by different kinetic steps results with those of the present study appears unwarranted. (10). It was proposed that the dissociation of ADP from Our conclusions are strengthened by the agreement between actomyosin, which enables the rebinding of ATP to this measurements carried out on HMM samples prepared by complex (Scheme 1), could be a rate-limiting step in the controlled tryptic digestions of myosin and on chymotryptic shortening velocity in vertebrate muscle (31). If this were true, HMM selectively cleaved at loops 1 and 2. In both cases, the then the cleavage at loop 1, which is proximal to the nucle- inhibition of actin sliding velocities can be related to loop 1 Table 2. Mean velocities of actin in the in vitro motility assays and actin-activated ATPase for HMM selectively cleaved at loop 1 and loop 2 Motility, ,um/s Actin-activated ATPase HMM Species Mean velocity SD SEM Kin, ,iM Vmax, s- HMM 3.87 0.91 0.04 30 ± 3 13.3 ± 1.3 cL2-HMM 3.25 1.00 0.05 41 ± 4 10.8 ± 1.1 cLl-HMM 2.03 0.81 0.06 33 ± 3 11.7 ± 1.1 cL1,2-HMM 2.00 0.73 0.06 60 ± 6 11.1 ± 1.1 The velocities of more then 100 filaments were analyzed for each sample. Each experiment was repeated five times. The absolute values of mean velocities varied somewhat between different preparations, but the relative differences in the motility of the HMM species were the same in each experiment. Downloaded by guest on September 25, 2021 Biochemistry: Bobkov et al. Proc. Natl. Acad. Sci. USA 93 (1996) 2289 cleavage. The results of this study are also consistent with 4. Chaussepied, P. & Morales, M. F. (1988) Proc. Natl. Acad. Sci. several prior observations. These include the findings on the USA 85, 7471-7475. effects of (i) loop 2 cleavage on the Km values of actomyosin 5. Yamamoto, K. (1990) Biochemistry 29, 844-848. 6. Cheung, P. & Reisler, E. (1992) Biochem. Biophys. Res. Commun. ATPase (13, 28, 36), (ii) loop 2 sequence on actomyosin 189, 1143-1149. ATPase but not the in vitro motility of actin filaments (9), and 7. Warrick, H. M. & Spudich, J. A. (1987) Annu. Rev. Cell Biol. 3, (iii) the lack of enzymatic perturbation of actomyosin (Vma, 379-421. and Km values) by loop 1 cleavage (4, 13). These and other 8. Spudich, J. (1994) Nature (London) 372, 515-518. studies established the lack of direct correlation between 9. Uyeda, T. Q. P., Ruppel, K. M. & Spudich, J. (1994) Nature ATPase measurements in solution and the myosin motor (London) 368, 567-569. velocity in the in vitro motility assays. The loop hypothesis of 10. Umemoto, S. & Sellers, J. (1990) J. Biol. Chem. 265, 14864- a to that It 14869. Spudich (8) proposed solution problem. suggested 11. Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, that the structure and sequence of loop 1 regulate the motor M., Malik, F. & Vale, R. (1991) J. Cell Biol. 112, 1189-1197. velocity by influencing product release from the adjacent 12. Mornet, D., Pantell, P., Audermard, E. & Kassab, R. (1979) nucleotide-binding site and that loop 2 controls the actomyosin Biochem. Biophys. Res. Commun. 89, 925-932. ATPase, presumably via changes in weak actomyosin binding. 13. Mornet, D., Bertrand, P., Pantell, P., Audermard, E. & Kassab, Obviously, the separation between these two functions is R. (1981) Biochemistry 20, 2110-2120. incomplete. In the absence of factors that compensate for the 14. Balint, M., Wolf, L., Tarcsfalvi, A., Gergely, J. & Sreter, F. A. inhibition of weak actomyosin binding (methylcellulose), such (1978) Arch. Biochem. Biophys. 190, 793-799. 15. Godfrey, J. & Harrington, W. F. (1970) Biochemistry 9, 886-893. an inhibition also results in the loss of motion. 16. Spudich, J. A. & Watt, S. (1971) J. Biol. Chem. 246, 4866-4876. The results of this work support the main assumption of the 17. Weeds, A. G. & Pope, B. (1977) J. Mol. Biol. 111, 129-157. loop hypothesis on the importance and the different roles of 18. Margossian, S. S. & Lowey, S. (1982) Methods Enzymol. 85, loops 1 and 2 in myosin. It is interesting to speculate about the 55-72. mechanism by which loop 1 may control the velocity of the 19. Mocz, G., Szilagyi, L., Chen Lu, R., Fabian, F., Balint, M. & myosin motor. At least in the present work, the changes in the Gergely, J. (1984) Eur. J. Biochem. 145, 221-229. motor function are not reflected in ATP turnover rates by 20. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 21. Homsher, E., Wang, F. & Sellers, J. R. (1992)Am. J. PhysioL 262, acto-Sl and the rates of eADP release from acto-Sl. Other C714-C723. observations, showing an 80% inhibition of actin motility by 22. Rosenfeld, S. S. & Taylor, E. W. (1987) J. Biol. Chem. 262, MgsADP (11) despite similar off rates ofADP and eADP from 9994-9999. acto-Sl (37), also indicate that ADP release is not necessarily 23. Phan, B. C., Faller, L. D. & Reisler, E. (1993) Biochemistry 32, limiting the velocity of actin filaments. This suggests that loop 7712-7719. 1 may regulate the myosin motor velocity via a different step 24. Taylor, E. W. (1977) Biochemistry 16, 732-740. in the crossbridge cycle. For example, it has been suggested 25. Johnson, K. A. & Taylor, E. W. (1978) Biochemistry 17, 3432- that the lower shortening velocity of muscle fibers in the 3442. 26. Ueno, H. & Harrington, W. F. (1984) J. Mol. Biol. 180, 667-701. presence of CTP may be explained by a slower rate of 27. Maita, T., Hayashida, M., Tanioka, Y., Komine, Y. & Matsuda, dissociation of actomyosin by CTP (38). Alternatively, loop 1 G. (1987) Proc. Natl. Acad. Sci. USA 84, 416-420. may affect efficiency of coupling between catalytic events at 28. Yamamoto, K. (1991) J. Mol. Biol. 217, 229-233. the nucleotide-binding site and the conformational changes on 29. Hynes, T. R., Block, S. M., White, B. T. & Spudich, J. A. (1987) Si which produce motion. These possibilities may be examined Cell 48, 953-963. in kinetic studies with appropriate loop 1 mutants (8) which 30. Lowey, S., Waller, G. S. & Trybus, K. M. (1993) J. Biol. Chem. would offer a greater range of motor velocity changes than that 268, 20414-20418. 31. Siemankowski, R. F., Wiseman, 0. W. & White, H. D. (1985) achieved in the present work. Proc. Natl. Acad. Sci. USA 82, 658-662. 32. Marston, S. B. & Taylor, E. W. (1980) J. Mol. Biol. 139,573-600. We thank Dr. L. Faller for making his stopped-flow facility available 33. Taylor, E. W. (1991) J. Biol. Chem. 266, 294-302. to us. This work was supported by U.S. Public Health Service Grant 34. Ohichi, T., Hozumi, T. & Higashi-Fujime, S. (1993)J. Biochem. AR22031, National Science Foundation Grant MCB 9206789, and an (Tokyo) 114, 299-302. American Heart Association Greater Los Angeles Affiliate fellowship 35. Toyoshima, Y.-Y., Kron, S. J., Niebling, K. R. & Spudich, J. A. (to A.A.B.). (1988) Biophys. J. 53, 238a (abstr.). 36. Botts, J., Muhlard, A., Takashi, R. & Morales, M. F. (1982) 1. Ma, Y.-Z. & Taylor, E. W. (1994) Biophys. J. 66, 1542-1553. Biochemistry 21, 6903-6905. 2. Rayment, I., Rypniewsky, W. R., Schmidt-Base, K., Smith, R., 37. Rosenfeld, S. S. & Taylor, E. W. (1984) J. Biol. Chem. 259, Tomchik, D. R., Bennig, M. M., Winkelmann, D. A., Wesenberg, 11920-11929. G. & Holden, H. M. (1993) Science 261, 51-58. 38. White, H. D., Belknop, B. & Jiang, W. (1993) J. Biol. 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