Pharmac.Ther.Vol. 55, pp. 95-148, 1992 Printed in Great Britain. All rights reserved

0163-7258/92 $15.00 © 1993PergamonPress Ltd

Associate Editor: J. G. CORY

ACTIN MEDIATED REGULATION OF MUSCLE CONTRACTION JOSEPH M. CHALOVICH* Department of Biochemistry, East Carolina University, School of Medicine, Greenville, NC 27858-4354, U.S.A. Abstract--Striated and smooth muscles have different mechanisms of regulation of contraction which can be the basis for selective pharmacological alteration of the contractility of these muscle types. The progression in our understanding of the tropomyosin-troponin regulatory system of striated muscle from the early 1970s through the early 1990s is described along with key concepts required for understanding this complex system. This review also examines the recent history of the putative contractile regulatory proteins of smooth muscle, caldesmon and calponin. A contrast is made between the actin linked regulatory systems of striated and smooth muscle.

CONTENTS 1. Introduction 2. Hydrolysis of ATP by Myosin and Actomyosin 3. Regulation by Tropomyosin-Troponin 3.1. The tropomyosin-troponin complex 3.2. Conformationai changes of tropomyosin-troponin 3.3. Result of tropomyosin movement 3.4. The steric blocking model 3.5. The allosteric model of regulation 3.6. The nature of the regulated transition 3.6.1. Evidence from fiber studies 3.7. Challenges to the allosteric model of regulation 3.8. Weak and strong binding myosin crossbridges 3.9. Stabilization of weak and strong binding states 4. Complications of Actin Mediated Regulation 4.1. Multiple step binding of myosin to actin 4.2. Actin-tropomyosin conformationai states 5. Summary: Regulation by Tropomyosin-Troponin 6. Smooth Muscle Actin Binding Proteins 6.1. Caldesmon 6.2. Calponin 7. Conclusion Acknowledgements References

95 97 103 103 105 107 108 110 111 115 117 119 121 122 122 127 131 132 132 134 135 135 135

1. I N T R O D U C T I O N The ability to sustain directed movement is a fundamental criterion of life. We have, in our bodies, several types of 'molecular motors' which can convert chemical energy into the mechanical *Supported by Grants AR35216 and AR40540-01AI from the National Institutes of Health. Abbreviations--S-1, subfragment 1 of myosin--the globular catalytic region; HMM, heavy meromyosin-the large fragment of myosin with 2 catalytic sites; LC1, light chain 1; LC2, light chain 2; LC3, light chain 3; EDC, 1-(ethyl-3°(3-dimethylamino)propyl)carbodiimide; pPDM, N,N'-p-phenylenedimaleimide; ATP~S, adenosine 5'-(~,,-thio)triphosphate; ~ATP, 1, N6-ethenoadenosine triphosphate; ~ADP, 1, N6-ethenoadenosine diphosphate. 95

96

J.M. CHALOVICH

Rod "I= chymotrypsin+

EDTAT J~4t

Myosin ~Lchymotryps~4- MgCl2

HMM

~

Lldld

FIG. 1. Basic structure of skeletal muscle myosin and its subfragments. Myosin (center) consists of a coiled coil tail (the coiling is not shown) which terminates in two globular head regions shown as solid ellipsoids. These globular heads or S-1 regions bind to actin and hydrolyze ATP. Each head contains two noncovalently bound polypeptide light chains: one LC2 (indicated by an asterisk), which can be phosphorylated and either LC1 or LC3. Myosin is insoluble as a result of aggregation of the tail region into thick filaments. Soluble fragments suitable for kinetic and binding studies can be produced by digestion of myosin with chymotrypsin. Digestion in the presence of MgCI 2 produces insoluble light meromyosin (LMM) and the soluble catalytically active fragment heavy meromyosin (HMM). Digestion in the absence of Mg2÷ produces an insoluble rod fragment and two S-1 fragments. Note that one light chain, LC2, is lost during the preparation of S-1 with chymotrypsin; the remaining light chain is either LC1 or LC3. energy of movement. The most obvious of these molecular motors is myosin which works in conjunction with the protein actin. Actomyosin, the complex of actin and myosin, is responsible for the movement of skeletal, cardiac and smooth muscles. The basic mechanism by which force or movement is produced by these three types of muscles is thought to be similar. However, important differences exist in the regulatory apparatus of these three muscle types. Thus a detailed knowledge of these regulatory systems provides the possibility of selectively intervening in the function of a single muscle type. Because muscle contraction requires the participation of both actin and myosin it is reasonable that regulatory systems might be directed toward both of these proteins. Both actin-linked and myosin-linked regulatory systems do exist. Actin-linked regulation involves muscle specific actin binding proteins which can control either the binding of actin to myosin or affect the ability of actin to participate with myosin as a cofactor in ATP hydrolysis. This type of regulation is common in both skeletal and cardiac muscle. The possibility also exists that a different type of an actin-linked regulatory system is operative in smooth muscle. Myosin-linked regulation in human muscle occurs by phosphorylation of the light chain components of myosin. This regulatory system is of greatest importance in smooth muscle and in actomyosin directed movement in nonmuscle cells. This review is limited to actin-linked regulation. The tropomyosin-troponin system of cardiac and skeletal muscle is heavily emphasized and caldesmon and calponin, of smooth muscle, are described briefly. The reason for this limitation is so that proper attention can be given to key concepts, such as ATP hydrolysis, weak and strong binding states of myosin and 'active' and 'inactive' forms of the actin filament. These concepts are important in all types of regulation but are most easily developed through a discussion of the tropomyosin-troponin system. The discussion of tropomyosin-troponin begins with the concept that Ca 2÷ binding to troponin causes changes in the troponin complex which ultimately change the interaction of tropomyosin with actin. Possible events by which this change in the tropomyosin-actin interaction can lead to

Actin mediated regulation of muscle contraction

97

FIG. 2. Model of actin decorated with tropomyosin (top) and tropomyosin alone (bottom). Each actin monomer is shown as consisting of two globular domains. Actin monomers are arranged in a helical array and tropomyosin can bind along each groove of the helix (tropomyosin is shown binding along one of the two grooves in the figure). A single tropomyosin molecule spans 7 actin monomers. The actin structure can be thought of as two actin strands twisted about each other with crossover points every 350-380/~ with about 13 actin monomers between these crossovers. Tropomyosin is a coiled coil of two helical subunits. Tropomyosin tends to associate in a head to tail fashion as shown in the bottom panel. Courtesy of Dr George Phillips. activation of contraction are then considered in the 'steric' and 'allosteric' models of regulation. Possible step(s) of ATP hydrolysis by actomyosin which are finally activated by C a 2 + are considered in Sections 3.9, 4.1 and 4.2. An understanding of regulation of contraction requires a physical picture of the proteins involved. The structure of the regulatory proteins will be introduced as needed. The key players in all of the following sections are myosin and actin, so their structures will be introduced here. The protein myosin is schematically illustrated in Fig. 1. The helical tail region of myosin is responsible for the formation of the thick filaments in muscles and causes the formation of insoluble myosin aggregates in low ionic strength solution. The catalytically active regions of myosin are the two globular heads. Each head contains two noncovalently associated polypeptides known as light chains. One of these, light chain 2 (LC2), can be phosphorylated. The other light chain present on each head can be of two isoforms, light chain 1 (LC1) or LC2. The two myosin heads of a single myosin molecule are called a crossbridge since this unit bridges the gap between the thick myosin filaments and thin actin filaments in muscle. To avoid the problems associated with aggregation of myosin, solution studies are often done using proteolytic fragments of myosin known as heavy meromyosin (HMM) or subfragment 1 (S-l) (Cooke, 1989). The structures of these fragments are illustrated in Fig. 1. The detailed 3-dimensional structure of S-1 is not completely known but the work is in progress (Winkelmann et al., 1991). The protein actin exists in two forms, monomeric or globular and polymeric or fibrous. As shown in Fig. 2, fibrous actin has a helical structure (Milligan et al., 1990; Holmes et al., 1990; De Rosier, 1990; Egelman and DeRosier, 1991). Recently the atomic structure of a complex of actin and deoxyribonuclease I has been determined to 3 A resolution (Kabsch et al., 1990). Actin structure and properties are reviewed elsewhere (Carlier, 1991; Pollard, 1990; Vandekerckhove, 1990; Korn, 1982).

2. H Y D R O L Y S I S O F ATP BY MYOSIN A N D A C T O M Y O S I N An important concept in understanding movement and the regulation of movement is the mechanism by which myosin and actomyosin hydrolyze ATP. Myosin can, by itself, hydrolyze ATP at a low rate. It is only when myosin binds to actin that ATP is hydrolyzed rapidly and that movement is possible.

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Initial attempts to decipher the mechanism of ATP hydrolysis by myosin were done in the simple case where actin is absent. Numerous studies (Bagshaw et al., 1974; Koretz and Taylor, 1975; Taylor, 1977; Inoue and Tonomura, 1973; Chock and Eisenberg, 1979) on the kinetics of ATP hydrolysis by myosin (M) and its subfragments, have lead to the schemes similar to the following one: M + T ~ M - A T P ~ M * A T P ~ M * * A D P P i ~ M + ADP + Pi Myosin binds to ATP in at least two stages beginning with a weak collision complex and undergoing a conformational change to form a stable M*ATP complex. This complex, M*ATP, has an increase in myosin tryptophan fluorescence (indicated by the *). The binding to ATP is followed by a rapid cleavage of the phosphate anhydride bond which gives a further increase in fluorescence. This hydrolysis step is much faster than the subsequent steps in the reaction so that there is an initial rapid formation of a mixture of M-ATP and M-ADP-Pi (Lymn and Taylor, 1970). In assays using acid to quench the reaction, this myosin-bound Pi is released and gives rise to an apparent 'initial burst' of phosphate. The slow steady state rate of ATP hydrolysis by myosin was thought to be due to the slow release of products from the myosin (Taylor et al., 1970). The release of both Pi and ADP may be multiple step processes but these are not shown in the scheme. Rate constants for the steps in this scheme have been tabulated elsewhere (Woledge et al., 1985). In the presence of magnesium ions, actin causes a large acceleration in the rate of ATP hydrolysis by myosin (Maruyama and Watanabe, 1962). However, detailed analyses of the kinetics of actin activation are not possible in this system because of the complex intermolecular reactions known as superprecipitation occur during this process (Maruyama and Gergely, 1962). The kinetics of ATP hydrolysis by actomyosin are also difficult to study because they do not follow simple Michaelis-Menten kinetics. That is, the values of Vmaxand KATPaseare not unique but differ at high and low actin concentration ranges (Pope et al., 1981; Strzelecka-Golaszewska et al., 1979). The reasons for this complex behavior are not completely understood. Most studies of actomyosin ATPase activity have been done using a soluble proteolytic subfragment of myosin such as HMM or S-1 as described earlier (Eisenberg and Moos, 1967; Yagi et al., 1965; Sekiya et al., 1967). The ATPase activities of these subfragments follow Michaelis-Menten kinetics, with respect to actin concentration and the rates of ATP hydrolysis are 10 times faster than with myosin and are more representative of the rates in a working muscle (Eisenberg and Moos, 1968). Most of the studies described in this review deal with myosin subfragments. Another interesting experimental approach is to use synthetic myosin 'minifilaments' which have many of the advantages of myosin subfragrnents while still retaining the entire myosin molecule (Reisler, 1980). Early experiments with HMM showed that high degrees of activation of ATPase rates occurred under conditions where there was very little binding of actin to HMM (Leadbeater and Perry, 1963; Eisenberg and Moos, 1967). It was later suggested that the binding of ATP to HMM greatly weakened the binding of HMM to actin (Eisenberg and Moos, 1968). Although the binding of HMM-ATP to actin was weak, the rate of ATP hydrolysis increased in a hyperbolic manner as the free actin concentration was increased. Analysis of the actin concentration dependence using the Michaelis-Menten approach allowed the assignment of values of Vmaxand KAxPasefor this reaction. The large stimulatory effect of actin on rate was assumed to be due to an increase in the rate of product release. Lymn and Taylor subsequently observed that the addition of ATP to an actin-HMM complex caused a very rapid dissociation of HMM from actin (Lymn and Taylor, 1971). This rate of dissociation, at low protein concentrations, was about 10-fold faster than the phosphate burst or hydrolysis step. In fact, the rate of dissociation may be on the order of 5000 sec ~at 20 °C (Millar and Geeves, 1988). Lymn and Taylor suggested that hydrolysis of ATP to ADP + Pi occurred on the free HMM after dissociation from actin was completed. The M**DPi complex could then reassociate with actin, at a rate proportional to the actin concentration (Fig. 3A). Lymn and Taylor also showed directly that the rate of product dissociation from the acto-HMM-ADP-Pi complex was much faster than from the HMM-ADP-Pi complex. The Lymn-Taylor model was later modified as a result of the observation that a lower free actin concentration was required to reach 50% of the maximum ATPase (KAxw~)activity than required

Actin mediated regulation of muscle contraction

®

M

~--~

If AM

M-ATP¢

I,

t fsst ~

99

.ADP . . . . • M, ADP...~,_ M.pi • . . . .

It

If

ADP.. .AOP... A.M'p i ~ A ' M

A'M-ATP

slow

slow ADP ._~ . ,ADP . . . . • M,ADP

® M

._~

1t AM

M.pi I

""

t fast _.~

~

"'Pi,

" ....

It

It

A-M-ATP

M-ATP

M

AM

M-ATP._~

, ADP~_ ,ADP A.M.Pil I . - ~ A'M

slow K M ADP ~ M.AD P_.._91~ M.AD P K11

"Pil

~-

"Pl,"

....

.ADP ADP KIO ADP K12 A'M-ATP _ . ~ A'M. Pil _ . ~ A'"'., Pill ~ A'M" "1-"..~

FIG. 3. Three models of ATP hydrolysis by myosin in the presence of actin from the 1970s through the 1980s. M is myosin or a myosin subfragment and A is actin in these schemes. Broken arrows are used to indicate a transition with a very low probability of occurrence. (A) The Lymn-Taylor model (Lymn and Taylor, 1971). (B) The refractory state model (Eisenberg et al., 1972; Mulhern and Eisenberg, 1976). (C) The 6 state model (Stein et al., 1979; Stein, 1988). Subscripts to Pi are used to indicate proposed conformational changes. to reach 50% binding of S-1 to actin in the presence of ATP (gbinding) (Eisenberg et al., 1972).* However, if product release were rate-limiting as in the Lymn-Taylor model, the apparent KATPase would equal the observed dissociation constant. To explain this and subsequent similar observations (Mulhern and Eisenberg, 1976; Marston, 1978; Wagner and Weeds, 1979) it was necessary to postulate that either the hydrolysis step (that is, the burst) was rate limiting or that an additional step was present in the cycle. Because the burst was observed to be a rather fast process Eisenberg and coworkers proposed that a rate limiting conformational change occurred just prior to product release (Stein et al., 1979, 1984, 1981; Stein, 1988). The model proposed by Eisenberg and coworkers contained an additional step shown in Fig. 3B as M-ADP-Pi(I) to M-ADP-Pi(II) and referred in older literature as M - A D P - P i ( R ) to M - A D P - P i ( R ) . t This 'refractory state' model allowed the essential features of the ATPase cycle to be maintained while still allowing for a high rate of ATP hydrolysis with little binding of S-1 to actin. That is, because the transition from state I to state II is slow, state I, which was thought to be refractory toward binding to actin, is the most heavily populated state. The presence of this extra state allows for successful modeling of both the difference between KATPase and Kbinding and the large phosphate burst when myosin is bound to actin. An alternate *All binding constants and Michaelis constants are expressed here as association constants. tThe suffixes (R) and (N) refer to the refractory and nonrefractory states, respectively. The term refractory was used to indicate that this state M'°DPi was refractory toward binding actin. It was later shown that this state did, in fact, bind to actin and the nomenclature was changed to (I) and (II).

100

J.M. CHALOVICH

model, in which state II is omitted would be possible if the difference between KnTPasoand Kbi,ai,~ were small (Rosenfeld and Taylor, 1984) and if the size of the phosphate burst decreased substantially when myosin bound to actin (Tesi et al., 1990; Belknap et al., 1992). There continues to be a discussion over whether the hydrolysis step is rate-limiting or whether an additional step, following hydrolysis is rate limiting. One significant change in the original Lymn-Taylor model for which there is agreement regards the irreversible nature of the dissociation of acto-S-1 by ATP. Several studies suggested that myosin could bind to actin even in the presence of ATP. Marston postulated reversible binding of S- I - A T P to actin as a way of explaining biphasic curves of ATPase rate as a function of actin concentration (Marston, 1978). Another indication of binding of myosin to actin, in the presence of ATP, comes from the actin concentration dependence on the inhibition of resynthesis of ATP from ADP and Pi (Sleep and Hutton, 1980). Actin inhibits the resynthesis of ATP since actin binds more tightly to M - A D P than to M - A T P . To fit their experimental data, Sleep and Hutton assumed binding constants of 2 x 104 M- 1 for the binding of M - A T P to actin and 4 × 106 M- I for the binding of S-1-ADW ° to actin. Although not direct measures of binding, these experiments did suggest that S-1-ATP could bind to actin, although less tightly than does S-I-ADP. The first direct measurement of the binding of S-1-ATP to actin was done by Stein et al. (1979). They observed that upon mixing S-1-ATP with actin in a stopped-flow spectrophotometer that there was a very rapid formation of an acto-S-1 complex. The rate of this process was faster than the phosphate burst completed within 5 msec of mixing at 15 °C. At the 1 m u ATP used in this experiment the binding of ATP to S-1 or acto-S-1 and the subsequent dissociation of S-I from actin both have rate constants near 1000 sec-~ (Lymn and Taylor, 1971; Johnson and Taylor, 1978). Therefore, the binding observed was due to the rapid equilibrium binding of S-1-ATP to actin to form A-S-1-ATP. The binding observed by Stein et al. was due not only to S - I - A T P but also to S-1-ADP-Pi. This is because S-1-ATP and S-1-ADP-Pi have an equilibrium constant near 1 at low temperatures (Sleep and Taylor, 1976) and this equilibrium is rapidly established (Wagner and Weeds, 1979; Sleep and Boyer, 1978). In support of this, Stein et al. observed that the level of binding remained constant before and after the initial burst of ATP hydrolysis. Furthermore, the same initial level of binding was observed if S-1 was mixed with actin + ATP or if S-t was preincubated with ATP (giving the S-1-ADP-Pi state) prior to mixing with actin. This showed that both S-1-ATP and S-1-ADP-Pi bound rapidly to actin with similar binding constants. The amount of this binding increased with the actin concentration and extrapolated to 100% binding at infinite actin concentration.* Following complete hydrolysis of the ATP there was a further increase in binding since the S-1-ADP complex bound to actin more tightly than the S - i - A T P complexes. This binding of S-1-ATP and S-1-ADP-Pi to actin is much weaker than the interaction that occurs in the presence of ADP. In addition to showing that ATP did not lead to complete and irreversible dissociation of actomyosin, Stein et al. (1979) showed that ATP hydrolysis could occur at high actin concentrations where the S-1 remained bound to actin throughout the cycle (Fig. 3C). If cleavage of the terminal phosphate anhydride bond (the burst) could occur only when myosin was not bound to actin then the rate of ATP hydrolysis should be inhibited at very high actin concentrations when the binding of S - I - A T P to actin was favored. However, such inhibition was not observed (Chalovich et al., 1984b). The hydrolysis of ATP without mandatory dissociation of S-I from actin was confirmed by Mornet and coworkers (Mornet et al., 1981). They observed that S-1 could be covalently crosslinked to actin using a carbodiimide reagent, 1-(ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC). The covalent acto-S-1 complex hydrolyzed ATP at a rate similar to the Vmaxobtained at very high aetin concentrations. Thus ATP hydrolysis does not require that myosin detach from actin (see also Stein et al., 1985; Biosca et al., 1985; Rosenfeld and Taylor, 1984). It will be seen *Stein et al. (1979) were able to observe binding by using high protein concentrations and low ionic strength. In a muscle the actin and myosin are arranged in a lattice and binding reactions do not require diffusion of two proteins together but more nearly resemble an isomerization reaction. As a result, the interaction of myosin with actin in muscle occurs as if the actin concentration were very high; that is around 1 mM or higher (Brenner et al., 1986b). This very high 'effective actin concentration' cannot be duplicated in solution and so, low ionic strength conditions are used to favor association reactions.

Actin mediated regulation of muscle contraction

~

ATP

~

ADP Pl

..,

~

tADP

101

very slow .... ~ADP

.,.

J-Pi

,~ K9 "~'

~.

f .~Fi

~K10

I

weak ~ I ,

I

,

FIG. 4. A crossbridge model of ATP hydrolysis and force production. Actin is represented as a string of circles and a myosin S-1 group is shown projecting from the backbone of a myosin thick filament. Transition Kl0 is thought to represent the force producing event shown as a change in the binding of myosin to actin as Pi is released.

later that this weak binding of S-1 to actin during ATP hydrolysis is a key point in the regulation of contraction. The model shown in Fig. 3C shows the nondissociating pathway of ATP hydrolysis. Note that this discussion of ATP hydrolysis is, by no means, complete. Rather those concepts essential for the discussion of regulation have been emphasized. For a more complete discussion of the mechanism of actomyosin ATPase activity, the reader is directed to several excellent reviews (Hibberd and Trentham, 1986; Stein, 1988; Homsher and Millar, 1990). A plausible description of the contractile cycle, based on the description of Eisenberg and Greene (1980), is given below (see Fig. 4). Myosin or actomyosin binds rapidly to ATP and the terminal phosphoanhydride bond is cleaved in an equilibrium reaction. This results in formation of an equilibrium among a number of 'weak binding' crossbridge states including M-ATP, AM-ATP, M - A D P - P i and AM-ADP-Pi. Evidence has been presented that M-ADP-Pi and A M - A D P - P i exist in two conformational states and that the transitions from M-ADP-Pil to M A D P - P i . and from AM-ADP-Pi] to A M - A D P - P i , are slow steps. Little is known about the properties of the states M - A D P - P i . and A M - A D P - P i . . The release of products occurs next and, since this step is very slow for myosin alone, appreciable flux through the cycle occurs only through the transition AM-ADP-Pi to AM-ADP. The phosphate release step is very important since this step is implicated in force production (Hibberd et al., 1985; Pate and Cooke, 1989; Metzger and Moss, 1991). The binding of myosin to actin is thought to change dramatically with this phosphate release. The relaxed state is often depicted as a '90 °' configuration of the crossbridge (Reedy et al., 1965) and following activation and Pi release the binding is depicted as a '45 °' attached state (Pringle, 1967; Huxley, 1969). The force-producing change may not actually be a change from a 90 ° to a 45 ° attached state; the force-producing transition has not been identified. However it is convenient to express force production as such a change as shown in Fig. 4. A central concept to the rotating crossbridge hypothesis of contraction is that each chemical state of the myosin crossbridge (i.e. M-ATP, M-ADP-Pi, M-ADP, etc.) have a preferred type of interaction with actin. Thus it follows that the rate and equilibrium constants for all reactions involving binding of myosin to actin and all transitions between two actomyosin species are thought to vary with the type of attachment to actin, or strain on the crossbridge (Hill, 1974; Eisenberg and Hill, 1978). In a muscle fiber, a single crossbridge may be constrained by the other crossbridges in the fiber or by an outside force. Thus a single crossbridge may not always be at its preferred configuration of attachment. Thus myosin states containing bound ATP or ADP + Pi are most stable (least strained) at the '90 °' configuration while states containing ADP or no nucleotide are most stable (least strained) at the '45 °' configuration. The change in configuration from the 90 ° to the 45 ° configuration is thought to produce force or movement. Although this is the favored working hypothesis, proving that such a conformational change is the force producing event is one of the great challenges in muscle research. Following the release of Pi, the muscle is in a force producing state. If allowed to shorten, the crossbridges will move to their new stable state of 45 ° type attachment. To complete the cycle, ADP is released to form the AM complex which then binds to ATP forming, once again, A M - A T P but

102

J.M. CHALOVICH

now in a highly strained conformation. That is, while the A M - A T P state is most stable at a 90 ° attachment, it is now constrained in a 45 ° type attachment. This is an unstable situation. The rate of dissociation of M - A T P from A M - A T P is very fast and this negatively strained crossbridge can detach and reattach to another actin monomer in a more favorable orientation. It is interesting to note that M - A D P has been recently shown to attach and detach with actin, rather rapidly, in isometric muscle (Brenner, 1991). The implications of this observation are beyond the scope of the present review but are discussed in the report by Brenner (1991). Some care is necessary when attempting to compare kinetic measurements made in solution to kinetic measurements within a muscle fiber. Among other differences is that, in muscle, various transitions are dependent on the strain or 'angle' of attachment of myosin to actin; in solution no strain is possible and the myosin attaches to actin at its most favorable position (Hill, 1974; Eisenberg and Hill, 1978). Thus, the rate-limiting step in solution is always the same reaction step, whereas the rate-limiting step in muscle can change to a different reaction step depending on whether the muscle is contracting isometrically (no motion) or isotonically (with shortening). According to the 1957 crossbridge model of Huxley, crossbridges bind at a moderate rate ( f ) to actin, the force-producing conformational change occurs and the crossbridges detach rapidly (g2) at the end of this conformational change or power stroke (Huxley, 1957). During the conformational change, when the crossbridge is doing work, the rate of detachment (g~) is the slowest step. We now know that the crossbridge cycle is somewhat more complex than supposed at the time that this model was proposed. The rates of crossbridge attachment and detachment are now known to be too fast to be the transitions f and g. Rather, it is more proper to think o f f as the transition in the forward direction from the nonforce producing (i.e, M-ATP, AM-ATP, M-ADP-Pi, AM-ADP-Pi, etc.) to the force producing states (i.e. AM-ADP, AM); the reverse of these transitions,f_ is assumed to be very slow and is usually ignored. Similarly, g represents those processes which allow a return to the nonforce producing states, continuing in the forward direction and is not to be confused with f _ . Again, the reverse process, g is usually assumed to be very slow and is neglected. In terms of Fig. 4, attachment and detachment reactions occur in the vertical direction; the processes defined by f and g represent a cycle from (M-ATP + AM-ATP) to A M - A D P and continuing with release of ADP and finally rebinding of ATP to complete the cycle. Since the process described by ' f ' involves several sequential reaction steps it is common to refer to an apparent value o f f or fapp" The value Offapp in the fiber is similar to the rate constant for the rate limiting step of ATP hydrolysis in solution. This was shown by comparing the rate of force redevelopment of a lightly loaded shortening muscle following a quick restretch (Brenner, 1985) to the rate of ATP hydrolysis of myosin S-1 crosslinked to actin to maximize the ATPase rate (Stein et al., 1985; Mornet et al., 1981). The rate of these transitions was found to be similar in the fiber and in solution (Brenner and Eisenberg, 1986). When a fiber is contracting at maximum velocity the value of gapp is thought to be large. That is, the force-producing conformational change of myosin on the actin is allowed to occur without resistance and the gapp is thought to be very fast for negatively strained crossbridges (i.e. crossbridges at the end of the power stroke). In mechanisms such as that proposed by A. F. Huxley, the value ofgapp is dictated by the rate constant of detachment of myosin from actin (Huxley, 1957). This in turn is probably limited by the rate at which ADP is released from the myosin (ATP rebinding to actomyosin is very fast as is the subsequent detachment of myosin from actin). Siemankowski and White observed that at physiological temperature and ATP concentration, ADP release is slow enough such that the rate of dissociation of S-1 from actin could limit the rate of unloaded shortening in cardiac muscle (Siemankowski and White, 1984; Siemankowski et aL, 1985). A more extensive discussion of the dependence of rate constants on the displacement of myosin can be found elsewhere (Eisenberg et al., 1980; Eisenberg and Hill, 1978). The force, stiffness and ATPase activity of a fiber can be shown to be related to the values of fapp and gapp: Force = F = n *~'*fapp/(f~pp + gapp) Stiffness = S = n * ~*fapp/( lapp + gapp) ATPase = n * b *f app*gapp / ( f app "1- gapp)

Actin mediated regulation of muscle contraction

103

where n is the number of active crossbridges per half sarcomere (the natural contractile unit), and ~ are the mean force and stiffness, respectively, for a crossbridge in a force producing state and b is the number of half sarcomeres in a given fiber (Brenner, 1988).

3. REGULATION BY TROPOMYOSIN-TROPONIN 3.1. THE TROPOMYOSIN--TROPONINCOMPLEX It is clear from the previous section that ATP hydrolysis and movement require the active participation of both actin and myosin. That is, while myosin is capable of cleaving the terminal phosphate anhydride bond of ATP, it remains the function of actin to facilitate the release of the products of hydrolysis. Because of the important role played by both actin and myosin, modification of either protein could modulate the cycle of ATP hydrolysis. The first actin-linked regulatory system discovered is the relaxing factor from vertebrate striated muscle (Ebashi and Ebashi, 1964). This relaxing factor was found to be composed of tropomyosin, discovered earlier by Bailey (1948) and an additional complex of proteins called troponin (Ebashi and Kodama, 1965). Tropomyosin exists as a dimeric ~ helical coiled coil (Caspar et al., 1969; Cohen and SzentGyorgyi, 1957). Tropomyosin binds to F-actin with a stoichiometry of 1 tropomyosin: 7 actin monomers (Bremel et al., 1972; Spudich and Watt, 1971) and lies along the groove of the actin helix (Hanson and Lowy, 1963; O'Brien et al., 1971). Figure 2 shows a model of tropomyosin and the actin-tropomyosin complex from the crystal structure of tropomyosin at 15 /~ resolution (Phillips et al., 1986). The crystal structure of tropomyosin has recently been determined to 9/~ resolution by X-ray diffraction (Whitby et al., 1992). The primary structure of tropomyosin has 14 groups of clustered acidic amino acid residues which are thought to be involved in binding to actin (McLachlan and Stewart, 1976; Parry, 1975). These acidic residues can be grouped into 2 sets of quasiequivalent sites, ~ and fl (Hitchcock-DeGregori and Varnell, 1990; McLachlan and Stewart, 1976). Only the ~ sites are thought to be regular enough to be important in actin binding (Phillips et al., 1986). However, genetic deletion analysis has been used to argue for 14 quasiequivalent actin binding sites (Hitchcock-DeGregori, 1992). The other component of the regulatory complex, troponin, was shown to consist of at least two polypeptides (Hartshorne and Mueller, 1968; Schaub and Perry, 1969). Three component polypeptides isolated from the troponin complex were later shown to be required for full Ca 2÷ regulation (Greaser and Gergely, 1971). These three components were named troponin I, troponin T and troponin C. In the presence of tropomyosin, troponin I inhibits ATP hydrolysis by actomyosin and the addition of troponin C neutralizes the effect of troponin I. Upon the further addition of troponin T, the system becomes fully Ca2÷-sensitive. It was later shown that the complex of troponin I and troponin T inhibits ATP hydrolysis even in the absence of tropomyosin (Eisenberg and Kielley, 1974). Troponin C prevents the inhibition of ATPase activity in either the absence of troponin T or tropomyosin but restores full regulation in the presence of the other components. Troponin T was thought to prevent Troponin C from neutralizing the inhibitory effect of TNT in the absence of Ca 2÷ Figure 5 illustrates the probable locations of the troponin components, tropomyosin and actin as viewed down the actin axis. The protein-protein contacts at high and low free [Ca 2÷] are shown. Another view of the actin-tropomyosin-troponin complex, which shows the location of tropomyosin and troponin within the actin helix is shown in Fig. 6. Readers are referred to other excellent reviews for greater details about these protein-protein interactions (Zot and Potter, 1987; Leavis and Gergely, 1984). Only an outline of the properties of the troponin subunits is given below. Troponin I binds to troponin C, troponin T and to actin (see Leavis and Gergely, 1984). Troponin I, alone can bind to actin in a 1:1 complex and inhibit ATP hydrolysis (Perry et al., 1972; Eisenberg and Kielley, 1974; Eaton et al., 1975). This inhibition is enhanced in the presence of tropomyosin and the stoichiometry of binding required for inhibition is reduced to 1 troponin I per 7 actin monomers, the same stoichiometry as the binding of tropomyosin to actin. A 21-residue cyanogen bromide fragment of troponin I, comprising residues 96 to 116 also inhibits actin

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°°

°

.:Tm;

; Tm',"

°°,

"Tin"

Fio. 5. Hypothetical view down the axis of an actin filament (A) showing the location of tropomyosin (Tm), troponin T (TnT), troponin I (TnI) and troponin C (TnC). The upper figure represents the state with two molecules of Ca 2÷ (c) bound to each troponin C at the Ca 2÷-specific regulatory sites. The other metal binding sites of troponin C probably contain bound Mg 2÷ (m). The broken circles, in the upper figure, indicate the proposed position of tropomyosin in the absence of Ca 2÷ . The bottom figure shows the proposed change in the troponin interactions when Ca 2÷ is removed from troponin C. Note particularly the proposed change in position of tropomyosin from its position at higher Ca 2÷ concentrations (shown as a broken circle in the lower figure). Drawn after an illustration given by Dr James Potter. activated ATPase activity (Syska et al., 1976; Wilkinson and Grand, 1978). A synthetic peptide of troponin I has been prepared which inhibits actomyosin ATPase activity; the activity of this peptide is enhanced by tropomyosin (Talbot and Hodges, 1979). In a later paper, Talbot and Hodges (Talbot and Hodges, 1981) varied the synthetic amino acid composition and showed that residues 105-114 are of particular importance for inhibition. Troponin T is an elongated molecule (Flicker et al., 1982) which binds to tropomyosin at two sites (White et al., 1987). The amino terminus of troponin T binds tropomyosin at 1/3 of the length of tropomyosin from its C-terminal end and extends beyond the C-terminal end and interacts with the adjacent tropomyosin; this can be seen in Fig. 6. Residues 1-70 of troponin T are implicated in the binding to the C-terminus and the overlap region of tropomyosin while residues 71-158 at the C-terminus and about 1/3 of the length of tropomyosin away from the C-terminus. Troponin T also binds to troponin I. In the presence of tropomyosin, troponin T can inhibit actomyosin ATPase (Chong et al., 1983). In the presence of tropomyosin, troponin I and whole troponin are equally potent inhibitors whereas twice the molar concentration of troponin T is required to reach the same level of inhibition. Troponin T does not bind to actin; it exerts its effects through some change in the tropomyosin. Troponin C reverses 50% or more of the inhibition of ATPase activity of troponin I, even in the absence of Ca: +; addition of Ca 2÷ totally reverses the inhibition. In the case of troponin T, troponin C does not affect the degree of inhibition unless Ca 2÷ is present and then about 50% reversal is achieved. Troponin C is a Ca 2÷-binding subunit. Troponin C binds to both the T and I subunits. As stated earlier, the troponin C - C a 2÷ complex reverses the inhibition of ATPase activity caused by the other inhibitory subunits. Troponin C has 4 Ca 2÷ binding sites (Potter and Gergely, 1975; Ikemoto et

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FIG. 6. Model of the regulated actin filament in the presence and absence of Ca 2+ . As in Fig. 2, tropomyosin is shown in only one of the actin grooves. In contrast to Fig. 2, the troponin complex can be seen; it is shown in the presence of bound Ca 2+ (top) and absence of bound Ca 2÷ (bottom). Notice the slight change in position of tropomyosin relative to the actin monomers as well as to the troponin. This is a good illustration of how subtle the conformational change is which triggers contraction. Courtesy of Dr George Phillips. al., 1974) and it is the binding to the weak binding sites that is responsible for regulation (see Zot and Potter, 1987). Troponin C resembles calmodulin somewhat in structure (Babu et al., 1985; Herzberg and James, 1985; Sundaraligam et al., 1985). Notably, troponin C has the EF hand Ca 2+ binding site characteristic of the family of calcium binding proteins including calbindin and parvalbumin (Kretsinger, 1980). Fragments of troponin C have been characterized which are capable of restoring Ca 2+ sensitivity to the inhibition of ATPAse activity by troponin I alone or in the presence of troponin T (Weeks and Perry, 1978; Grabarek et al., 1981).

3.2. CONFORMATIONAL CHANGES OF TROPOMYOSIN TROPONIN The details of the events that occur between the binding of Ca 2+ to troponin C and the production of force in striated muscle are not known. However, this is an area of intensive investigation and an outline of the molecular events is being formed. We consider below some changes that occur upon the binding of Ca :+ to troponin C. An advance in the understanding of the troponin complex came with the determination of the crystal structure of troponin C (Herzberg and James, 1985, 1988; Sundaralingam et al., 1985). Figure 7 shows a representation of the crystal structure obtained in the absence of Ca 2+ In general, binding of Ca 2+ to the low affinity, regulatory binding sites (I and I1) are thought to form a structure similar to that of the structural, high affinity Ca2+-binding sites (III and IV). This results in a movement of helices B and C away from helices A and D. This could cause exposure of more potential sites of contact with troponin I and alter the binding of troponin C to troponin. Recent evidence suggests that troponin C may be folded so that the N-terminal and C-terminal regions are in close contact unlike the structure shown in Fig. 7 (Swenson and Fredricksen, 1992).

106

J. M.

CHALOVICH ÷

I

/oo III

-

FIG. 7. Proposed 3D structure of troponin C (Herzberg and James, 1985; Sundaralingam et al., 1985). Helicies labeled A through G are shown by cylinders. The two high-affinity Ca 2+ binding sites (III and IV) are located at the COOH-terminus and the low affinity, regulatory sites (I and II) are at the N-terminal region. Only two molecules of Ca 2÷ were seen in the crystal (shown as circles in regions III and IV). The bold letters X and Y represent Gln48 and Gln 82. The bond between Gln 48 and Gln 82 was introduced experimentally to test models of structural change (see text). Two experimental tests of this model have been done. Grabarek et al. (1990) substituted cys residues for Gln 48 and Gln 82 (X and Y in Fig. 7, respectively). Formation of a disulfide bridge between these two cys residues created a bridge between the B and C helices which prevented the opening of a cavity for the binding of Ca 2+ to site II. This had the effects of reducing the Ca 2+ affinity, of locking the troponin C into its inhibitory configuration at both high and low Ca 2+ and reducing the binding to troponin I. Normal activity of the troponin C was restored by reducing the disulfide bridge. Fujimori et al. (1990) took a similar approach to this problem. They produced mutants where either Glu 57 (helix C) or Glu 88 (helix D) were replaced by lys residues. These mutations caused small decreases in the affinity for Ca z + particularly for the regulatory sites. The formation of maximum tension of skinned rabbit muscle fibers whose troponin C was exchanged with these mutant troponin C's was shifted to higher concentrations of Ca 2+. The decreased regulatory function of these mutants was proposed to be due to the formation of a salt bridge in the mutants (either LysSV-Glu 88 or Glu57-Lys88). Such a salt bridge would conceivably stabilize the troponin C having no Ca 2+ bound to the specific regulatory sites. The changes in the structure of troponin C, upon binding to Ca ~+, result in changes in a number of other protein interactions. These many changes have already been described in detail (Leavis and Gergely, 1984; Zot and Potter, 1987). In general, all of the interactions among the 3 troponin subunits become stronger, in the presence of Ca 2+, while interactions of the troponin subunits with actin and tropomyosin become weaker. As shown in Figs 5 and 6, the tighter binding of troponin C to troponin I, in the presence of Ca z +, occurs at the loss of direct interaction between troponin ! and both actin and tropomyosin. In addition, the binding of troponin T to tropomyosin is weakened. The idea that has emerged is that, at low Ca 2+, the troponin complex keeps tropomyosin away from its preferred location of binding to actin. The binding of Ca 2+ to troponin C allows tropomyosin to bind to actin in its preferred location. The precise nature of all of the changes that occur with Ca 2+ binding are not yet known but there is a great deal of work being done on this area. This last event, the movement of tropomyosin on actin, was historically the first change detected in the actin-tropomyosin-troponin complex. The observation of a change in the binding of tropomyosin to actin came from careful observations of X-ray diffraction and optical diffraction

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patterns of muscles and muscle proteins. Upon activation of a muscle there is no obvious change in the gross structure of the actin itself seen by X-ray diffraction studies (Elliott et al., 1967; Huxley and Brown, 1967). However, one reflection, the 2rid layer line, was observed in active muscle but not in relaxed muscles (Huxley, 1972). This 2nd layer line reflection was shown to be due to the tropomyosin molecule (O'Brien et al., 1971). Huxley confirmed that the 2nd and 3rd layer lines were strengthened by tropomyosin and proposed that tropomyosin forms a continuous strand along each of the two grooves of actin. An increase in the 2nd layer line and decrease in the third layer line was observed upon activation of frog skeletal muscles (Huxley, 1972; Vibert et al., 1972) as well as smooth and molluscan muscles (Vibert et al., 1972). These data were explained by models by which the tropomyosin molecule moved into a different position on the actin filament in the presence of Ca 2÷ (Spudich et al., 1972; Parry and Squire, 1973; Haselgrove, 1972; Huxley, 1972). In these models, actin was assumed to be spherical and tropomyosin was approximated by a cylinder. Tropomyosin was assumed to always be closely bound to actin and the movement of tropomyosin was restricted to an arc of constant radius from the center of the actin filament. With this simple model, the observed changes in the diffraction pattern could be reproduced by a change in position of tropomyosin to one closer to the actin groove in active muscle. The range of positions of tropomyosin in both the presence and absence of Ca 2÷ depended on further assumptions which differed in each model. In Haselgrove's model, tropomyosin was thought to move from an angle of 50° to one of 70 ° upon activation but a reasonably good fit could also be obtained if in the active state the tropomyosin was between 60 ° and 90 °. A recent computer generated model of the actin-tropomyosin-troponin (Fig. 6) clearly shows the proposed structural change of actin-tropomyosin-troponin. The change in tropomyosin position was thought to be smaller in molluscan 'catch' or vertebrate smooth muscle than in vertebrate striated muscle (Parry and Squire, 1973). The binding of Ca 2÷ alone, was sufficient for the observed changes in X-ray reflections in vertebrate skeletal muscle since muscles pulled out of overlap, so that no contact between myosin and actin is possible, give the same changes in reflections (Haselgrove, 1972). The change in position of tropomyosin was confirmed by 3-dimensional reconstructions of electron micrographs using optical diffraction methods (Wakabayashi et al., 1975). In this study, whole muscle was not used. Rather, to simplify the system, the structure of actin-tropomyosin (used as a model of the active filament) was compared to the structure of actin-tropomyosintroponin I-troponin T (used as a model of the inactive filament). It will be shown later that full activity of the actin-tropomyosin-troponin complex requires the binding of S- 1 to actin. Therefore the changes observed may not reflect a change from total inhibition to total activation. This is also a consideration in fiber studies where the fiber is pulled out of overlap and in most solution studies. As stated earlier, the change in position of tropomyosin on actin probably results from the Ca2÷-induced changes in the troponin complex. Phillips et al. suggested that in the presence of Ca 2÷, tropomyosin interacts favorably with actin primarily through the ct sites. In the presence of Ca 2÷ the troponin subunits interact strongly with each other and weakly with actin and tropomyosin (Phillips et al., 1986). Upon removal of Ca 2÷ an increased strength of binding of troponin to actin and tropomyosin occurs at the expense of a displacement of tropomyosin from its stable ~ site mediated binding to actin. In relaxed muscle, tropomyosin may be weakly and dynamically attached to actin. The question that arises is how this change in interaction of tropomyosin to actin regulates striated muscle contraction. 3.3. RESULT OF TROPOMYOSIN MOVEMENT The previous discussion showed that the binding of Ca 2+ to troponin C ultimately leads to a change in binding of tropomyosin to actin. This change in actin-tropomyosin must affect the interaction between actin and myosin in some way as to regulate contraction. Before considering evidence for different types of alteration of the actin-myosin interaction, it is helpful to consider what the possibilities for regulation are. This can be done by examination of the kinetic model shown in Fig. 4. One point that is apparent is that since tropomyosin-troponin binds to actin, only those processes which involve (a) the binding of myosin to actin, (b) a transition between two actomyosin states or (c) a transition between two actin conformational states (to be discussed later) J ~ 55/2--B

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J.M. CHALOVICH

can be directly regulated. Thus, none of the transitions occurring along the top line of Fig. 4 may be regulated. In contrast, a regulatory system which functions through the myosin molecule may conceivably affect any step in the cycle. Inhibition of the binding of M-ATP and M-ADP-Pi to actin would result in inhibition of ATP hydrolysis and contraction since both processes require binding of myosin to actin. Inhibition of the binding of M-ADP to actin, however, would not directly inhibit contraction since this occurs after the power stroke and after elimination of Pi. Regulation of the rate of transition between two actomyosin intermediates (bottom line of Fig. 4) can only be effective if that same transition is slow in the absence of actin. Inhibition of the rate of the burst step (M-ATP to M-ADP-Pi) would not be a very efficient regulatory event since the burst is equally fast when myosin is not bound to actin. Hydrolysis could then proceed by 'side-stepping' the unfavorable reaction. In contrast, regulation could occur by control of Pi release from AM--ADP-Pi to AM-ADP since phosphate release is very slow for detached crossbridges. Relaxed striated muscles are characterized by low force and low stiffness (low rigidity). Therefore, the most heavily occupied states of relaxed muscle must be detached states or weakly attached states that have rapid rates of dissociation (see Section 3.8). For example, inhibition of the rate of ADP release would not be a good candidate for regulation since this would result in the accumulation of the tightly bound actin-myosin-ADP complex resulting in a rigid muscle. Therefore, the most reasonable candidates for regulation are the binding of 'weak' crossbridges to actin and the kinetic transitions associated with Pi release from attached crossbridges. The first possibility is an example of competitive inhibition whereas the second possibility would be a noncompetitive type of inhibition. Another point to be considered is whether regulation involves RECRUITMENT or a GRADED RESPONSE (kinetic modulation). That is, for a muscle that is operating at 50% maximum activity, are 50% of the crossbridges active (recruitment, see Podolsky and Teichholz, 1970) or are all of the crossbridges 50% active (graded response or kinetic modulation, see Julian, 1969). For tropomyosin-troponin regulation this is equivalent to asking whether each group of seven actin monomers acts rather independently and can be 'active' or 'inactive' irrespective of its neighboring actin groups or whether all groups of seven actin monomers are in one of many possible states of activation. Recent mechanical studies suggest that the latter possibility is correct, that is activation of striated muscle by Ca 2÷ is due to kinetic modulation (Brenner, 1988). 3.4. THE STERICBLOCKINGMODEL Both competitive and noncompetitive mechanisms were recognized at the time of the discovery of the change in position of tropomyosin on actin (Wakabayashi et al., 1975). However, it was generally assumed, that the movement of tropomyosin competitively inhibited the binding of myosin to actin (Parry and Squire, 1973; Haselgrove, 1972; Huxley, 1972). This model is called the steric blocking model of regulation. Steric blocking is also a special case of recruitment since the inability of myosin crossbridges to bind to actin would result in a decrease in the number of crossbridges which are able to go through the contractile cycle. There were several reasons for assuming a steric blocking mechanism. First, as stated earlier, the type of conformational change in actin expected for an allosteric modulation was not observed upon activation, at least in early studies. Thus an allosteric change in the actin as a result of changes in tropomyosin position was considered unlikely (Wakabayashi et al., 1975). This is an important point in distinguishing the steric blocking model from other models of regulation. That is, in the steric blocking model, actin is considered to be static and tropomyosin is responsible for controlling access of myosin to the actin sites. A second indication of steric blocking came from studies done in the early 1970s which suggested that bound myosin crossbridges overlapped the tropomyosin binding sites on actin, in the absence of Ca 2+ (Moore et aL, 1970; DeRosier and Moore, 1970). Localizing the position of S-1 and tropomyosin on the actin filament in a muscle is no easy task as subsequent studies showed. Following the work of Moore et al. (1970), which supported a steric blocking mechanism, Seymour and O'Brien (1980) observed that the tropomyosin and myosin heads bind to opposite sides of the actin filament where steric blocking would be impossible. In the next year, Taylor and Amos (1981) provided a new interpretation which placed the myosin and

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FIG. 8. View down the axis of an actin filament (A) showing the positions of tropomyosin in the 'relaxed' position (open broken circle) and in the 'active' position (circle T). Two proposed locations of the S-1 region of myosin are shown on the actin. Steric blocking of the binding of myosin would be possible in position S-1a (Taylor and Amos, 1981) but not in position S- 1b (Seymour and O'Brien, 1980). Allosteric regulation would be possible in either position. tropomyosin in close proximity once more where steric blocking could occur. The latter two orientations of tropomyosin and S- 1 are illustrated in Fig. 8. It is clear that steric blocking involves more than movement of tropomyosin; the movement must overlap the binding site of S-1 sufficiently to prevent myosin binding in relaxed muscle. The reader is referred to an interesting and brief summary of the changing position of tropomyosin and myosin (Squire, 1981). A further complication, for steric blocking, came from the observation that a single S-1 could be covalently crosslinked to two actin molecules with the crosslinking agent E D C (Mornet et al., 1981). This result was supported by subsequent image reconstructions (Amos et al., 1982; Wakabayashi and Toyoshim~, 1981). In this type of model, the tropomyosin could be located between the two myosin binding sites. This provides a tempting speculation that the tropomyosin movement could inhibit the transition from one type of bound state to another type of bound state rather than by inhibiting binding. At the time of postulation of the steric blocking model, the tropomyosin molecule was assumed to be rigid and statically bound to actin in either of two positions. However, detailed studies of the crystal structure of tropomyosin suggest a very different picture. The persistence length of tropomyosin (the longest distance over which molecular motion are linked) has been estimated to be 2-4 molecular lengths (Phillips and Chacko, 1992). Similarly, electric birefringence measurements of skeletal tropomyosin may be modeled as a semi-flexible rod with a persistence length of 150 nm or 3.6 molecular lengths (Swenson and Stellwagen, 1989). This persistence length places an upper theoretical limit on the cooperativity through tropomyosin, in the absence of bound S-I. Moreover, this persistence length is a manifestation of the high flexibility of tropomyosin (Phillips and Chacko, 1992). Tropomyosin cannot be treated as a rigid rod as assumed in early models of steric blocking. It is thought, for instance, that even if one actin molecule, of a group of 7 covered by tropomyosin, were blocked from binding to myosin, other actins within the group could bind to myosin. Thus, a strict steric blocking may not be possible. As the structures of S-I, actin and tropomyosin are becoming known to a higher level of resolution, it is becoming clear that there is not a complete overlap of S-1 and tropomyosin binding sites on actin. Milligan et al. (1990) recently used image analysis cryoelectron microscopy to localize the binding sites of S-1 and tropomyosin on the reconstituted protein complex. In activating conditions, S-1 was seen to form its major contact with the outer domain of actin and extend toward the inner domain of actin near the tropomyosin binding site. S-1 also binds to the top of the outer domain of an adjacent long pitch monomer in agreement with earlier cross linking studies (Marianne-Pepin et al., 1985; Mornet et al., 1981). A prediction of models of regulation which involve movement of tropomyosin on the actin filament is that this movement precedes force development in the muscle. To test this prediction,

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Huxley and coworkers measured the time course of structural changes in muscle during activation using synchrotron radiation (Kress et al., 1986). The second actin layer-line, thought to be due to reflect the position of tropomyosin, increases following stimulation and reaches half maximum intensity after about 17 msec. The equatorial 11 reflection, which monitors the attachment of myosin crossbridges to actin, reaches its half maximum intensity at 25-30 msec. Finally, tension reaches its half maximum intensity at 40--50 msec after stimulation. In agreement with earlier studies, the change in position of tropomyosin was not dependent on the binding of crossbridges to actin although a model in which Ca 2+ gives only partial activation with further activation occurring upon crossbridge binding could not be ruled out. These data strongly suggest that the change in orientation of the tropomyosin molecule is an important event in regulation since it precedes both an increase in crossbridge attachment and tension development. Huxley and Kress (1985) suggested that crossbridges first bind to actin in a 'preforce-generating' state whose binding is controlled sterically by the tropomyosin position on actin. Force was thought to be produced following transition into a 'force-generating' state. Brenner (1990) showed that the data of Huxley and Kress can be explained without postulating the existence of the 'preforce-generating' states but by assuming that force is produced from a transition from weak binding to strong binding states (Brenner, 1990). In this alternate model the affinity of weak binding crossbridges is only slightly altered by Ca 2÷ and the major regulatory event is the rate of transition from weak binding to strong binding states. The hypothesis that regulation of contraction does not occur by steric blocking is explored later in this chapter. Not all of the changes in the actin-tropomyosin-troponin complex precede myosin crossbridge attachment. Ishii and Lehrer have used the excimer fluorescence of rabbit skeletal tropomyosin labeled a t c y s 19° with N-(1-pyrenyl)-iodoacetamide to monitor tropomyosin-actin conformational changes (Ishii and Lehrer, 1990, 1991). No change in excimer fluorescence occurs until after myosin crossbridges bind to actin; Ca 2÷ itself has little effect (Ishii and Lehrer, 1991). It is also noteworthy that the X-ray diffraction changes attributed to movement of tropomyosin are seen in fibers pulled out of overlap so that there is no binding of myosin to actin. However, further changes in ATPase activity (Bremel et al., 1972; Murray et al., 1982; Nagashima and Asakura, 1982; Pemrick and Weber, 1976; Dancker, 1992; Cande, 1986; Meeusen and Cande, 1979) and in fiber contractility (Schnekenbuhl et al., 1992; Swartz et al., 1992) occur when crossbridges bind to the actin filament. Therefore, the observed X-ray changes may not reflect the total regulatory response. 3.5. THE ALLOSTERICMODEL OF REGULATION Although the steric blocking model is simple and intuitively pleasing, it does not readily explain all the experimental data. Weber and colleagues observed that under some conditions, tropomyosin-troponin can actually stimulate the rate of ATP hydrolysis above that of actin alone (Bremel et al., 1972; Murray et al., 1982, 1980). In the steric blocking model, stimulation of ATPase activity would be impossible since actin by itself was considered to be maximally active and tropomyosin could have either no effect (in Ca 2+) or inhibit ATP hydrolysis (at low free Ca 2+) by blocking the binding of myosin. Tropomyosin alone can also inhibit or potentiate (Williams et al., 1984; Lehrer and Morris, 1984; Eaton, 1976) the rate of actin-activated ATP hydrolysis. This potentiation is also evident in smooth muscle tropomyosin which is not normally associated with troponin (Williams et al., 1984; Lehrer and Morris, 1984; Chacko and Eisenberg, 1990). These observations are more readily explained by a model in which tropomyosin alters the conformation of actin in an allosteric fashion. It is interesting to note that maleimidobenzoyl-G-actin (Bettache et al., 1990) stabilized with phalloidin has a potentiated ATPase rate in the absence of tropomyosin or troponin (Miki and Hozumi, 1991). This raises the possibility that actin alone can be stabilized in a more active or less active state suggesting that conformational changes in actin are important. Other effects of tropomyosin on ATPase activity are most easily explained by postulating conformational changes in the actin caused by tropomyosin. Rabbit skeletal actin does not activate the ATPase activity of Limulus myosin whereas skeletal actin-tropomyosin does activate the ATPase (Lehman and Szent-Gyorgyi, 1972). Also, skeletal muscle tropomyosin produces partial inhibition of acto-HMM ATPase activity and this inhibition is increased by the addition of troponin I (Eaton et al., 1975; Wilkinson et al., 1972). Therefore, the effects of tropomyosin and

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tropomyosin-troponin may be graded. It is possible to explain a graded inhibition by assuming that tropomyosin can adopt a continuum of positions which block the binding of myosin to varying degrees. However, there is no way to obtain enhancement of the rate over that with actomyosin alone by such a mechanism. Furthermore, the binding of S-1 to actin is actually enhanced by tropomyosin (Eaton et al., 1975; Eaton, 1976). Several experimental approaches have also given indication of a change in actin structure during activation. Yanagida and Oosawa (1980) measured the changes in polarization of fluorescence of the fluorescent ADP analog 1, N6-ethenoadenosine diphosphate (eADP) following its incorporation into F-actin. Single muscle fibers were extracted to remove myosin, tropomyosin and troponin (Yanagida and Oosawa, 1978) and eADP was then incorporated into the actin. Tropomyosin and troponin, but not myosin, were then added back to the fibers. The fluorescence polarization of the resulting fibers was Ca2+-sensitive. The angle of the base plane of eADP changed at pCa 2÷ of about 6 indicating a conformation change in the actin or a change in orientation of the actin monomers. The actin filaments were also more flexible in the presence of Ca 2+. The effect of tropomyosin-troponin on actin structure was also examined by polarized fluorescence of phalloidin-rhodamine-labeled F-actin in extracted fibers (Dobrowolski et al., 1988). The binding of myosin S-1 to such extracted fibers caused a change in orientation of the probe and an increase in the flexibility of actin thought to represent activation of the actin filament (Galazkiewicz et al., 1987). Yagi and Matsubara (1989) obtained evidence from X-ray diffraction of frog semitendinosis muscle fibers that an actin conformational change accompanied activation. The frog fibers were highly stretched so that interaction of myosin with actin was impossible. Upon activation, the equator, the second layer line at 1/18 nm-1 and the 5.9 nm layer line integrated intensity increased while the intensity of the first layer line at 1/36 nm- ~decreased by 48%. The first actin layer line partially overlaps the first myosin layer line at 1/43 nm-i. However, the changes were thought to be due to actin since the myosin layer line of highly stretched muscle does not change (Huxley et al., 1980; Yagi and Matsubara, 1980). The various changes in intensity could not be fitted by a model assuming the Egelman and DeRosier 2-domain structure of actin (Egelman and De Rosier, 1983) and treating tropomyosin as a cylinder with two positions on the actin filament even if the mass of troponin T (now known to be extended along the tropomyosin) were considered. Therefore, Yagi and Matsubara (1980) concluded that an additional conformational change in actin must occur. Kress et al. (1986) also observed that the 5.9 nm actin layer line changes simultaneously with changes in tropomyosin diffraction. The change in 5.9 nm layer line occurred even in overstretched muscles although to a lower extent. Tropomyosin movement, itself, should not contribute to a change in this layer line. Poppet al. (1991) have recently shown that by moving the small domain of actin, it is possible to duplicate the X-ray diffraction pattern of the actin-tropomyosin complex. These authors suggested that the thin filament conformational changes, seen during activation, are not due to tropomyosin alone but might also involve changes in actin structure. 3.6. THE NATURE OF THE REGULATED TRANSITION The key postulate of the steric blocking model is that the binding of myosin-ADP-Pi to actin is inhibited in the absence of Ca 2+. The first attempt at measuring the effect of Ca 2÷ on the binding of myosin-S-1 to actin-tropomyosin-troponin in solution was done using a stopped-flow device (Chalovich et al., 1981). It had been shown earlier that the weak binding of S-1-ADP-Pi to actin could be measured by observing the rapid increase in turbidity due to the binding of S-1 to actin in the presence of ATP (Stein et al., 1979). Rapid measurement of binding was required because the binding of S-1-ADP to actin is much tighter than the binding of S-1-ADP-Pi to actin and in the presence of Ca 2+, the rate of Pi release is very fast and could cause erroneously tight binding. The stopped-flow method was applied to the measurement of binding of S- 1 to actin-tropomyosintroponin in the presence of ATP in both the presence and absence of Ca 2+. Although C a 2+ increased the rate of ATP hydrolysis 25-fold, it resulted in only about a 2-fold increase in the binding constant Kbindi,g.This surprising result did not support the steric blocking model but rather indicated that the change in conformation of the actin-tropomyosin-troponin complex acted

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allosterically to regulate the rate of a process that occurred after myosin bound to actin. Shortly thereafter, another method of measuring the binding of S-1-ATP + S-1-ADP-Pi was developed which was based on the separation of free and bound S-1 species by rapid sedimentation in an ultracentrifuge (Chalovich and Eisenberg, 1982). This method gave excellent agreement with the stopped-flow method. Under conditions where 60% of the S-1 was bound to actin in either the presence or absence of Ca 2÷, the rate of ATP hydrolysis was more than 20-fold faster in the presence of Ca 2+. This showed that tropomyosin-troponin was capable of regulating the rate of some process that occurred after binding of S-1 to actin. These data alone, do not allow one to conclude that crossbridges are bound to actin in relaxed muscle. Such an extrapolation is difficult for several reasons: (1) Low ionic strength was used to promote binding so that the signal was large enough to measure accurately. In muscle the protein concentrations and the geometry favor binding so that it occurs even at much higher ionic strengths. These effects cause the 'effective' actin concentration, in muscle, to be quite high. In the rabbit psoas muscle, the effective actin concentration is 1.5~5.5 mM (Brenner et al., 1986b). (2) A single headed subfragment of myosin, S-I was used in the assays since myosin is insoluble at low ionic strength. (3) The geometry of binding is different in solution and in a fiber. Several efforts were taken to bridge the gap between these observations made in solution to a muscle fiber. Wagner and Giniger (1981) and Wagner and Stone (1983) confirmed the results of Chalovich et al. (198 l) on the binding of S-1 to actin-tropomyosin-troponin but observed different behavior with the two headed myosin fragment HMM. In the presence of Ca 2+, H M M bound with the same affinity as S-1. In the absence of Ca 2+ 33% of the H M M bound with the same affinity as in the presence of Ca 2+ but the remainder bound with a binding constant 1/20th of that in the presence of Ca 2+. Single headed H M M showed the same behavior. The greater sensitivity of binding of H M M and single-headed H M M was found to be due to the presence on intact LC2 which is lost during the preparation of S-1 (see Fig. 1). Phosphorylation of the LC2 did not further alter the binding. This should not be confused with phosphorylation of LC2 in smooth muscle myosin which appears to be required for actin activation of ATP hydrolysis. In this case phosphorylation is reported to have no effect on the binding of gizzard myosin (Sellers et al., 1982). In some types of smooth muscle, phosphorylation is reported to affect the KAvPaseand it has been argued that this is probably due to a strengthening of binding upon phosphorylation in these types of muscle (Wagner, 1986; Wagner and George, 1986). In a subsequent study, Wagner used urea gel electrophoresis to quantitate the amount of intact LC2 in the H M M and corrected the binding constant measured in the absence of Ca 2+ by assuming that those H M M molecules with a degraded LC2 (the degraded LC2 has an apparent MW of 17,000) will have the same binding constant as in the presence of Ca 2+ (Wagner, 1984). With these corrections, the Ca2+-dependence in binding became 10-fold. This difference was still too low to explain the difference in the rate of ATP hydrolysis meaning that tropomyosin-troponin affected a rate process. Furthermore, steady-state rate analyses indicated that the Vma× increased from 0.2-6.7 sec 1 (KATp,seincreased from 5 x 10 3 t o 5 × 10 4 M - I and Kbindingincreased from 4 x 10 3 t o 4 × 10 4 M - 1 ) , However, these data would suggest that a significant change in binding could occur in addition to changes in the rate of some process that occurs subsequent to the binding. Chalovich and Eisenberg (1986) also examined the binding of chymotryptic HMM, with intact LC2, to actin-tropomyosin-troponin. The binding constant, measured in the presence of Ca 2 +, was 3-fold higher than in the absence of Ca 2+ . Correction of these data for degraded LC2 brought the difference in affinity to 5-fold. A 'double binding' experiment was done to determine if there could be a larger difference in binding. In this experiment, the binding of H M M ATP to actintropomyosin-troponin was measured in the absence of Ca 2+. The supernatant, was presumably enriched in the fraction of H M M containing intact LC2 and having the weakest binding. This H M M was removed and the binding was repeated. Similar binding constants were obtained in both experiments indicating that the presence of LC2 does not have a dramatic effect on the Ca2+-sensitivity of binding. While there is a small difference in the binding of H M M to actin, the primary effect of tropomyosin-troponin was again to inhibit the rate of a process that occurred after binding was completed. An important consideration is whether a larger change in binding constant would occur at more physiological conditions, particularly at higher ionic strength. Inoue and Tonomura (1982)

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measured the binding of S-1 and HMM to actin-tropomyosin-troponin in both the presence and absence of Ca 2÷ as a function of ionic strength. At low ionic strength, they observed a 25% increase in the fraction of myosin bound to actin in the presence of Ca 2+. With increasing ionic strength the fraction of S-1 bound, decreased in parallel, in the presence and absence of Ca 2÷ . Calculation of binding constants from the data indicated that at 100 mM ionic strength there was less than a 6-fold difference in the binding constant in the presence and absence of Ca 2+. This result is in reasonable agreement with other studies. At 50 mM ionic strength, Chalovich and Eisenberg (1982) observed an increase in association constant of 1.5-fold while the ATPase activity increased 55-fold. El-Saleh and Potter (1985) studied the ATPase activity and binding in ATP at 134 mr~ ionic strength, 25 °. Under these conditions the association constant increased from 2.5 × 1 0 3 tO 2.7 x 103 M-1 upon the addition of Ca 2+. Therefore, even at ionic strengths close to physiological, tropomyosin-troponin and Ca 2÷ have relatively little effect on the binding of myosin subfragments to actin. A question that is always raised regarding binding measurements is whether the binding is specific. The interaction between S-1 or HMM and actin-tropomyosin-troponin saturates at a 1:1 ratio of S-1 to actin (Chalovich et al., 1981, 1983; Chalovich and Eisenberg, 1982) and it is correlated with a biological function which is the increase in rate of ATP hydrolysis (Chalovich et al., 1981; Chalovich and Eisenberg, 1982). This is particularly evident from a study in the effect of light chain composition on the binding of S-1 to actin. Small changes in the binding of these isozymes of S- 1 to actin are reflected in changes in the rate of ATP hydrolysis; that is a weakening of binding is associated with a weakening of the KATPase(Chalovich et al., 1984b). The ionic strength dependence of the binding of S-1-ATP is similar to that of the tight binding of S-1-AMP-PNP to actin (Chalovich et al., 1983). Additional evidence for specificity comes from the competition of S-1-ATP and HMM-ATP with caldesmon binding to actin in solution (Chalovich, 1988; Chalovich et al., 1990) and in single fibers (Brenner et al., 1991; Chalovich et al., 1991a,b). It is important to realize that the allosteric model of regulation does not preclude a change in myosin crossbridge binding to active muscle. Upon activation there is a large increase in the population of strong binding crossbridges. These strong binding crossbridges will, in fact, dominate many of the properties of the muscle because of their higher affinity and slower binding kinetics. Steady-state kinetics of the inhibition by tropomyosin-troponin also indicate that regulation is not a simple competitive steric blocking (Chalovich and Eisenberg, 1982). At very low ionic strength, the Vmax for ATP hydrolysis is about 18-fold greater in the presence of Ca 2÷ but there is only about a 2-fold increase in the KATpas~(association constant). A large change in the Vm~xis also seen at 50 mM (Chalovich and Eisenberg, 1982) and 134 mM (E1-Saleh and Potter, 1985) ionic strength. This large change in the Vm~x shows that in the inhibited state actin is rather ineffective in accelerating a step associated with product even when bound to myosin. Together with the observations that Ca 2+ has little effect on KAvPase and Kbinding this indicates that actin can exist in two or more conformational states which differ in their ability to catalyze product release. In a test of the ability of tropomyosin-troponin to modulate a rate process directly, two laboratories studied the effect of Ca 2+ on the ATPase activity of the S- 1-actin complex which was covalently crosslinked with EDC (King and Greene, 1985; Rouayrenc et al., 1985). The rate of ATPase activity was 20-fold higher in the presence of Ca 2+ than in the absence of Ca 2÷ although there was no possibility of dissociation of S-1 from the actin. This result was obtained only on preparations with low ratios of S-1 crosslinked to actin (King and Greene, 1985). At ratios of S-i to actin of 2:10 the rate in the absence of Ca 2+ was 92% of that at high Ca 2+. This was thought to be due to 'turning on' of the actin filament (see Section 4.2). Earlier kinetic studies with HMM had competitive type inhibition patterns (Eisenberg and Kielley, 1970; Parker et al., 1970). However, these studies did not involve the use of very high actin concentrations thus making an estimation of kinetic parameters difficult. Kinetic studies with tropomyosin, in the absence of troponin, also are indicative of regulation by an allosteric type of mechanism. Neither smooth nor skeletal muscle tropomyosin is a steric blocker or competitive inhibitor of actin activated S-1 ATPase activity (Sobieszek, 1982). Skeletal muscle tropomyosin, reduces the ATPase activity to about half of its initial value while smooth muscle tropomyosin activates the ATPase activity by up to 100%. Potentiation of ATPase activity, above the rate obtained with S-1 and actin alone, cannot be explained by steric blocking.

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Furthermore, the inhibition of ATPase activity by pure skeletal tropomyosin is due to both a 6-10-fold increase in the Kmpase (i.e. an increase in the apparent affinity) and a 6-10-fold reduction in the Vm,x. The inhibition was not of the competitive type predicted by the steric blocking mechanism. While these data do not indicate the mechanism of inhibition by the intact tropomyosin-troponin complex they do show that tropomyosin alone acts as an allosteric uncompetitive inhibitor or activator. This supports the role of conformational changes in actin in the regulation of muscle contraction. The preceding discussion dealt specifically with the regulatory system of vertebrate skeletal muscle; similar conclusions have also been reached using cardiac muscle regulatory proteins (Tobacman and Adelstein, 1986). Evidence has been presented that regulation involves inhibition of a kinetic transition aside from any changes in binding of myosin to actin that may occur. Chalovich and Eisenberg suggested that Pi release could be the step regulated by Ca 2+ by considering the reactions within the dashed box of Fig. 4 (Chalovich et al., 1981; Chalovich and Eisenberg, 1982). In this simple scheme, the equilibrium between M - A D P - P i and A M - A D P is path independent. That is, K9 x Kt5 = K~4 >