In the Motor Function of the Myosin Head (Heavy Meromyosin/Limited Tryptic Proteolysis/Velocity of Movement)

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.

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