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The Actomyosin Interaction and Its Control by Tropomyosin

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The Actomyosin Interaction and Its Control by Tropomyosin 2s Biophysical Journal Volume 68 April 1995 2s-7s The Actomyosin Interaction and Its Control by Tropomyosin Kenneth C. Holmes Max Planck Institute for Medical Research, 69120 Heidelberg, Germany An atomic model for actomyosin has been proposed the strongly bound state is created in a strained conformation (Rayment et al., 1993b) in which the actin and nucleotide from the weakly bound form. Because the strongly bound binding sites of S1 are linked by a cleft that may close on state has at equilibrium a different gross orientation to the binding to F-actin. The initial actomyosin contact probably actin filament (450) but is tethered by its attachment to the involves only part ofthe interface (a postulated stereospecific myosin filament to remain in the initial 900 position, in the weak interaction); the full rigor complex develops on closure new conformational state it is strained. Release of strain en- of the cleft. These states appear similar in properties to the ergy brings about the rowing-like sweep. A and R states described by Geeves (1992). Central to this kind ofcycle is the idea that the cross-bridge By analysis of the low-angle x-ray diffraction from rabbit must first bind in an unstrained conformation (weak) and muscle at nonoverlap with and without Ca21 (Poole et al., then undergo an isomerization to the strained (strong) form. 1994) the positions of tropomyosin can be derived. Com- Hill (1974) emphasized that the work done in sliding the bining these with the atomic model of actomyosin shows that filament, often referred to as the power stroke, should not with tropomyosin in the "off' state (no Ca21) the weak ste- occur between two states but within a state so that the stored reospecific binding would be inhibited. In the presence of energy can be smoothly given up into the muscle filament Ca21 the weak stereospecific interaction would be allowed. lattice. The process is modulated by the status of the nucle- However, the development of the full rigor interface would ne- otide binding site, in particular by the presence or absence cessitate a further small movement ofthe tropomyosin to permit of the y-phosphate. This is the heart of the contraction pro- the cleft to shut. These three states of the thin filament may be cess. A key question that we wish to address is a possible tentatively equated with the states "blocked," "closed," and structural basis for the weak-to-strong isomerization. "open" identified by McKillop and Geeves (1993). THE ISOMERIZATION BETWEEN A AND THE CROSS-BRIDGE CYCLE R STATES A widely accepted theory to explain the mutual sliding The initial attachment is weak in the sense that the bound of actin and myosin filaments is the cross-bridge theory of cross-bridge is in rapid equilibrium with the dissociated muscle contraction (see H. E. Huxley, 1969; A. F. Huxley, form. However, it should be stereospecific binding; other- 1974). This theory suggests that the sliding process is driven wise it would not be possible to provide a structural basis for by the myosin cross-bridges, which extend from the myosin the ensuing isomerization to the strong binding state. The filament and cyclically interact with the actin filament by a stereospecific weak binding to actin is required to enable rowing motion of the myosin head or part of the head as ATP (catalyze) the transition to the strong binding state. This se- is hydrolyzed. The Lymn-Taylor cycle for the hydrolysis of quence provides the necessary vectoriality to the process ATP by actomyosin (Lymn and Taylor, 1971) may easily be (Jencks, 1980). Without this property the cross-bridge would incorporated into the cross-bridge theory. This resulting bind unstrained in the strong state and nothing would happen. cycle has been elaborated by Eisenberg and Green (1980) to This scheme is not a unique paradigm for achieving a strained incorporate the fact that release from actin is not an obliga- bound cross-bridge. However, the two-step binding of S1 to tory step in the hydrolysis ofATP. In their cross-bridge cycle actin is in accordance with the biochemical data (Taylor, each sweep of an oarlike cross-bridge cycle is not linked 1991; Geeves, 1992) and provides a good starting model. rigorously to a biochemical event but the myosin cross- Geeves calls the two states the A and R states. The following bridge alternates between a weak binding conformation and quote from Geeves' paper gives the essential properties of a strong binding conformation, which differ in the structural this isomerization: way in which they interact with actin. In the muscle lattice "Studies of the interaction of actin and myosin S1 in solution have shown that the association reaction takes place in at least two steps. Initially the association is relatively weak to form a complex called the A state which can isomerise into the R state. The rate and equilibrium constants for the isomerisation have been measured and are shown to depend upon the nucle- Address reprint requests to Dr. Kenneth C. Max Holmes, Planck Institute otide bound to the Sl ATPase site; with ATP bound the A state is preferred for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. Tel.: but as ATP is hydrolyzed and the products are sequentially released then the 49-9221-486270; Fax: 49-6221-486437; E-mail: holmes@mpimf- complex gradually shifts to the R state. An extensive series of heidelberg.mpg.de. experiments ... have shown the isomerisation ... to be closely linked with the key events C) 1995 by the Biophysical Society in the ATP-driven contraction cycle: the conformational change can be 0006-3495/95/04/02s/06 $2.00 monitored by probes on either actin or the nucleotide; the isomerisation can Holmes The Actomyosin Interaction and Its Control by Tropomyosin 3s be perturbed by an increase in hydrostatic pressure, the actin-induced ac- separates the upper and lower 50K fragments. Examination celeration of the product release from myosin is coupled to the A to R of the acto-myosin interface suggests that there are poten- isomerisation; tropomyosin may control actin and myosin interaction by three of interaction with actin: an ionic controlling the isomerisation step and finally pressure perturbations of con- tially types initially tracting muscle fibres shows there to be a close coupling between the isom- interaction involving the disordered 50-20K loop; secondly, erisation of acto-Sl and the force generating event of muscle contraction" a stereospecific interaction involving hydrophobic residues (Geeves, 1992, p. 63). that we associate with the weak A state; and thirdly, a strengthening of this interaction by the recruitment of more THE LOW IONIC STRENGTH INTERACTION MAY hydrophobic interactions allowed by the coming together of BE A PRE-WEAK STATE the 50K upper and lower fragments, which could produce the strong rigor interaction. Although the weak state we discuss here is conceptually The segment between residues 626 and 647 (50/20Kjunc- similar to the weak state discussed by Eisenberg and Green tion), which is disordered in the three-dimensional structure, (1980) it may not be the same as the weak states found at low contains five lysines and nine glycines (depending on the ionic strength (Brenner et al., 1982) since it has been shown, species). However, these lysine residues are protected from e.g., by Pollard et al. (1993) that at low ionic strength the proteolysis in the presence of actin, which suggests that while cross-bridges take up all possible orientations with respect to they are flexible in solution they are physically protected by the actin filament and appear therefore not to be stereospe- actin in the rigor complex (Mornet et al., 1979, 1981) or only cifically bound to actin. The interactions are presumably adopt a distinct conformation when bound in the actomyosin electrostatic in nature and appear to be incompetent to make interface. From their location the residues between 626 and the transition to the strong state (for discussion, see Wray 647 may interact with the negatively charged residues 1-4 on et al. (1988)). Such electrostatic weak states should be actin. It is expected that this interaction will be predomi- viewed rather as the cross-bridge making short-lived colli- nately ionic (five lysines in the loop and four carboxylic acid sion complexes with actin. Since the N-terminus of actin, groups near the N-terminus of actin) and therefore would be which is somewhat disordered, is highly negatively charged sensitive to ionic strength. The main contact of myosin with and the 50-20K myosin loop (see below), which is unstruc- actin involves the heavy chain segment from 525 to 552. This tured, is highly positively charged one could easily foresee consists of a helix that extends from 516 to 542, a loop from an interaction between these components ofactin and myosin 543 to 546, and a second helix from 547 to 558. The first leading to nonspecific complexes. helix, of which only part is in the actin binding site, contains a prominent bulge at residue 529. These two helices run at NAMING OF PARTS an angle of 10° to each other and are located at the end of the lower 50 K domain. These secondary structural elements Myosin S1 can be split by trypsin into three fragments are in close proximity to residues 341-354 and 144-146 of (Mornet et al., 1979) (25K, 50K, 20K from N- to C-termi- actin. This interface has a considerable hydrophobic com- nus), which were thought to be domains. Rayment et al. ponent. In addition residues 552 and 558 of myosin are close (1993a) show that these "domains" do not reflect structural enough to make contact with residues 40-42 in the actin divisions but rather that the trypsin-sensitive sequences lie in subunit below that, which forms the major interaction.
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