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Journal of Muscle Research and Cell Motility 24: 593–596, 2003.

Ó 2003 Kluwer Academic Publishers. Printed in the Netherlands.

What is the role of tropomyosin in the regulation of muscle contraction?

The detailed nature of the interaction between actin and molecule and it is difficult to understand how confor-
myosin upon which striated muscle contraction depends mational changes occurring in the troponin C, close to a
is not yet well defined nor is the precise mechanism by point where the tropomyosin interacts with an actin
which this interaction is regulated by the troponin monomer, result in the whole tropomyosin molecule
complex. Although molecular details of the calcium moving in an identical manner with respect to the actin
switch are now available from the high resolution surface extending over seven actin monomers.
structure of troponin C, we do not have similar Tropomyosin possesses two properties that are pre-
information about the other two components of the sumed to be of significance for its role in muscle and for
complex, troponin I and T. Despite this uncertainty this reason are usually incorporated in models of
tropomyosin has been considered for over 30 years to striated muscle regulation. (1) Its ability to interact with
have a well defined role in the regulation of contraction. actin. There are some unusual features in this interaction
It is of interest to reflect why this is so for the evidence is and the factors which determine it are complex. Tropo-
still circumstantial rather than direct. At the time the myosin can inhibit, activate or have no effect on the
steric hypothesis was proposed (Haselgrove, 1972; MgATPase of acto-heavy meromyosin, depending on
Huxley, 1972; Parry and Squire, 1973), the X-ray the ionic strength, the isoforms present, temperature, the
crystallographers and electron microscopists were mak- ratio of actin to myosin heads and the presence of other
ing the major contribution to the study of the structure myofibrillar proteins (see Perry, 2001, for review). (2) Its
and function of the myofibril. When changes in the X- ability to endow cooperative properties on the F-actin
ray diffraction pattern of the thin filament of intact filament. These are clearly shown by its effects on the
skeletal muscle during contraction were shown to be due behaviour of myosin MgATPase (Bremel et al., 1972)
to movement of tropomyosin in the actin groove it was and on the inhibitory properties of troponin I (Perry
logical to suggest that the former protein was involved et al., 1972).
in the regulatory process. This hypothesis postulating Interaction with actin is vital for a simple ‘blocking’
that tropomyosin ‘blocked’ the interaction of actin and mechanism from which cooperativity may or may not
myosin in resting muscle but not in the stimulated state, arise depending on the model proposed. It is now clear
was an elegant and plausible explanation of the obser- from several lines of evidence that in the ‘blocked’ or
vations. A special role for tropomyosin in the contractile resting state some interaction between actin and myosin
system seemed likely for at that time contractile tissue can take place, but not that which leads to the high rate
such as muscle and platelets appeared to be a unique of MgATPase associated with contraction (Chalovich
source of the protein. We now know that tropomyosin is et al., 1981; Stein et al., 1981; Resetar et al., 2002; see
widely distributed in vertebrate tissue invariably associ- Chalovich, 2002). Electron microscope reconstruction
ated with mature actin filaments. Muscle contains a studies of the position of tropomyosin on the actin
relatively large amount of the protein because the actin filament in the ‘blocked’ state (Vibert et al., 1997; Xu
thin filament system is a major component of the et al., 1999; Craig and Lehman, 2001) indicate that part
cytoplasm. It is questionable, had we known in 1972 as of the putative myosin binding site(s) is exposed. In
much about tropomyosin and its distribution as we consequence the original ‘blocking’ mechanism has been
know today, whether the steric hypothesis, giving it a modified to accommodate these observations.
unique role in muscle, would have been so widely The conclusion that tropomyosin ‘blocks’ the inter-
accepted. action of actin and myosin because in the resting state
It is beyond dispute that the tropomyosin molecule the position of tropomyosin on actin is close to those
moves on contraction in striated muscle but as yet the regions presumed to interact with myosin may not be
mechanism responsible is not understood. In the models justified. The interaction between actin and myosin is
incorporating a ‘blocking’ role for tropomyosin it is clearly complex with a number of contact points that
usually suggested the conformational changes known to may be involved in a coordinated manner and which are
arise when troponin C binds calcium are transmitted via not well defined. Most workers agree that on contrac-
the troponin complex (Pearlstone and Smillie, 1983; tion troponin moves with the tropomyosin but there is
Phillips et al., 1986, Heeley et al., 1987; Kobayashi still some uncertainty about its contribution to the X-
et al., 2000). These involve troponin T, which binds to ray data. It has, however, been distinguished from the
tropomyosin, with result that the latter protein changes latter in electron microscope reconstruction studies
its position in the groove of the actin filament to a ‘non- (Lehman et al., 2001). Nevertheless these studies have
blocking’ position. Tropomyosin is a rather flexible so far failed to prove it is the tropomyosin rather than
the troponin I that is binding to actin to prevent
* Tel.: +44-121-414-6930 interaction in the resting state. The troponin complex
extends alongside the tropomyosin occupying the C and troponin composed of one molecule each of
terminal half of the molecule with troponin I and C troponin I, C and T. If tropomyosin really ‘blocks’ a
located in a more central position with respect to the site on actin essential for activation it is easy to visualise
tropomyosin molecule (Perry, 1998). The experimental it lying alongside the actin filament close to the
evidence we have from in vitro studies suggests that in appropriate sites on each of seven actin monomers.
the absence of calcium i.e. in the ‘blocked’ state the The whole functional unit would be in effect under the
troponin I is bound to actin. As the calcium concentra- control of one tropomyosin molecule, as the steric
tion rises the affinity of troponin C for troponin I hypothesis implies. On the other hand, if as the more
increases markedly. The inhibitory peptide site of recent direct evidence suggests, troponin I interacts with
troponin I detaches from actin, troponin I binds to actin to produce the conformational change that pre-
troponin C and the actin monomers return to their vents interaction with myosin, only one actin monomer
ground state conformation in which they can activate (possibly two) will be directly involved. It is suggested
the myosin MgATPase. that in the presence of tropomyosin the conformational
Recent NMR and fluorescence studies (Patchell et al., changes imposed by troponin I on one actin monomer
2002; Patchell, Perry and Levine, unpublished), indicate are transmitted to monomers on either side in the
that peptides corresponding to sites on human cardiac filament. If these effects were transmitted to three
ß-myosin suggested from models as possible interaction adjacent actin monomers on each side of the troponin
sites, e.g. residues 398–414 and 622–646, bind to actin. I interaction site the whole functional unit would be
In the presence of troponin I, or the inhibitory peptide controlled by changes occurring on the actin monomer
derived from it, these myosin peptides dissociate from that binds troponin I (Figure 1). In view of the evidence
actin. The evidence indicates that the myosin peptides that possibly two actin monomers are involved at the
and the inhibitory peptide do not occupy identical sites site of interaction with myosin, one troponin I molecule
on actin and implies that interaction with troponin I may directly effect two actin monomers. If this were the
induces a conformational change in actin so that it is case the transmission of conformational changes be-
unable to interact with myosin in a manner that tween monomers would be aided.
activates the MgATPase. Tropomyosin did not block The structure of the double helical F-actin filament is
the binding of the myosin peptides; indeed it strength- stabilised by many interactions between monomers in
ened their affinity for actin. It also strengthened the the single filaments and between monomers in different
affinity of the inhibitory peptide for actin and in this way filaments of the duplex. It is likely that conformational
increased its ability to impose a conformational change changes imposed on single monomers will induce
on the actin. changes in adjacent monomers to maintain the double
There was no evidence that tropomyosin alone filament structure. The fact that troponin I molecules
inhibited the binding of any of the myosin peptides are located opposite each other on the adjacent actin
tested. These investigations must be extended to myosin filaments of the duplex (Figure 1) probably strengthens
subfragment 1 in the nucleotide bound states for further the effect of troponin I and the transmission of the
definition of the system. Nevertheless extrapolation conformational change along the actin duplex. The role
from the results obtained with peptide segments suggest of tropomyosin would be to stabilise the filament
that in the ‘blocked’ state interaction with these regions structure and thus facilitate the transmission of confor-
of the myosin molecule is prevented by a conformational mational changes along the filament. There are many
change imposed on actin by troponin I, not by the reports of the stabilising and stiffening effect of tropo-
‘blocking’ action of tropomyosin. myosin on the actin filament (Kawamura and Maru-
The functional unit of the thin filament consists of yama, 1970; Fujime and Ishiwata, 1971; Kojima et al.,
seven actin monomers, one molecule of tropomyosin 1994; Goldmann, 2000; Kuo-Kang et al., 2000). It is



Fig. 1. Diagrammatic representation indicating how conformational changes induced in actin by troponin I (TNI) could be transmitted to
neighbouring monomers in the thin filament (thin arrows). For the purpose of representation the actin duplex is uncoiled. Position of
tropomyosin indicated by a thick black line in resting muscle and a hatched line in contracting muscle.
clear that on binding, tropomyosin imposes some effect clear that conformational changes imposed on actin by
on the structure of actin for in its presence the affinities the unique inhibitory protein of striated muscle, tro-
of troponin I inhibitory peptides, myosin peptides ponin I, are of particular significance for regulation.
(Patchell et al., 2002), troponin (Wegner and Walsh, Movement of tropomyosin also occurs in smooth
1981) and myosin (Bremel et al., 1972; Eaton, 1976) are muscle although its location in the actin groove is
increased. different to that in striated muscle (Lehman et al., 1996).
If the conformational changes in actin are transmitted Myosin and particularly actin are strongly conserved
along the filament, changes will occur in the distribution proteins and despite the differences in the regulatory
of residues on its surface. Some of these residues will be system in smooth muscle, the role of tropomyosin in
involved in the binding to tropomyosin and the fibrous relation to actin would be expected to be similar to that
molecule will have to adjust its position along its whole in striated muscle. The difficulties in ascribing a ‘block-
length on the F-actin surface for energetic consider- ing’ role for tropomyosin in smooth muscle regulation
ations. The interaction between actin and tropomyosin are discussed elsewhere (Marston et al., 1998).
is rather unusual, certainly different from that between If the role of tropomyosin is to facilitate the trans-
actin and troponin I where specific sites are involved mission of conformational changes between actin mono-
(Grabarek and Gergley, 1987; Levine et al., 1988). mers the difficulties in explaining its role in all types of
Tropomyosin interacts with different actin monomers muscle disappear. Such a general role makes good
via each of the quasi-equivalent regions which although functional sense for actin-binding proteins with different
similar are not identical in sequence. The distance roles are widely distributed as are actin filaments. Actin
between the carbon atoms of actin and tropomyosin in filaments are built up of a large number of subunits
the model of the complex proposed by Lorenz et al. usually much more abundant than the functional
(1995) is about 10 Å which implies that the interaction is proteins that bind to them. It is biologically efficient
mainly ionic as the effects of magnesium and potassium for the conformational effects induced by the binding
chloride on complex formation suggest. With tropomyo- proteins on the actin filament to be transmitted to the
sin floating as it were in the actin groove its position is actin monomers with which they are not in contact.
determined largely by ionic interactions. Conformation- The tropomyosin molecule polymerised as a continuous
al changes on the surface of the actin monomer filament in each of the two grooves of the actin duplex is
probably involving both hydrophilic and hydrophobic ideally located for this role. The proposed mechanism
side chains would lead to a change in the position of the of function implies that the role of tropomyosin is
tropomyosin molecule. not a special case unique to muscle but merely an
The role of tropomyosin in inducing cooperativity example of the role played in all actin filament systems.
into the actin filaments is illustrated by the use of in vitro It is suggested that striated muscle is regulated by a
systems of actomyosin and troponin I. Addition of specialised allosteric switch applied by troponin I on
tropomyosin converts the effect of troponin I to produce actin and facilitated by the general properties of
maximum inhibition of the MgATPase from a molar tropomyosin.
ratio of troponin I to actin of approximately 1:1 to I am grateful to Val Patchell, Barry Levine, Emil
approximately 1:7 (Perry et al., 1972; Lehrer et al., Toescu and Joe Chalovich who have made helpful
1997; Geeves et al., 2000). Thus in the presence of comments on this article.
tropomyosin and troponin I, but in the absence of the
other components of the troponin complex, no actin S.V. Perry
monomer in the functional unit is able to fully activate Department of Physiology
the myosin MgATPase. The potentiation of the inhi- Division of Medical Sciences
bitory effect of the intact molecule of troponin I is also Medical School
obtained with 21 and 12 residue peptides (Syska et al., University of Birmingham
1976; Talbot and Hodges, 1981). It is difficult to Edgbaston
envisage a mechanism that enables such small peptide Birmingham B15 2TT
regions of troponin I to displace tropomyosin into an UK
inhibitory position along the whole of its length as a
‘blocking’ role would demand. A more plausible expla-
nation would be that tropomyosin facilitates the trans- References
mission of the effect of the conformational change
Bremel RD, Murray JM and Weber A (1972) Manifestations of
imposed by troponin I peptide on actin to all the cooperative behaviour in the regulated actin filament during actin-
monomers in the functional unit. activated ATP hydrolysis in the presence of calcium. Cold Spring
Until the atomic details of the actomyosin interface Harbor Symp Quant Biol 37: 267–265.
and nature of the interaction of the troponin complex Chalovich JM (2002) Regulation of striated muscle contraction: a
discussion. J Muscle Res Cell Motil 23: 353–361.
with it have been defined at high resolution it is not
Chalovich JM, Chock PB and Eisenberg E (1981) Mechanism of action
possible to be certain about the role of tropomyosin in of troponin–tropomyosin inhibition of actomyosin ATPase activ-
the regulatory process. In this discussion attention has ity without inhibition of myosin binding to actin. J Biol Chem 256:
been focussed on striated muscle where it is becoming 575–578.
Craig R and Lehman W (2001) Cross bridge and tropomyosin Levine BA, Moir AJG and Perry SV (1988) The interaction of
positions observed in native interacting thick and thin filaments. J troponin-I with the N-terminal region of actin. Eur J Biochem 172:
Mol Biol 311: 1027–1036. 389–397.
Eaton BL (1976) Tropomyosin binding to F-actin induced by myosin Lorenz M, Poole KJV, Popp D, Rosenbaum G and Holmes KC (1995)
heads. Science 192: 1337–1339. An atomic model of the unregulated thin filament obtained by X-
Fujime S and Ishiwata S (1971) Dynamic study of F-actin by ray fiber diffraction on oriented actin–tropomyosin gels. J Mol Biol
quasielectric scattering of laser light. J Mol Biol 62: 251–265. 246: 108–119.
Geeves MA, Chai M and Lehrer SS (2000) Inhibition of actin–myosin Marston SB, Burton D, Copeland O, Fraser A, Gao Y, Hodgkinson P,
ATPase activity by troponin I and IC: relationship to the thin Huber PA, Levine B, El-Mezgueldi M and Notoriani G (1998)
filament states of muscle. Biochemistry 39: 9345–9350. Structural interactions between actin, tropomyosin, caldesmon and
Goldmann WH (2000) Binding of tropomyosin–troponin to actin calcium binding protein and the regulation of smooth muscle thin
increases filament bending stiffness. Biochem Biophys Res Commun filaments. Acta Physiol Scand 164: 401–414.
276: 1225–1228. Parry DAD and Squire JM (1973) Structural role of tropomyosin in
Grabarek Z and Gergely J (1987) Troponin–I binds to the N-terminal muscle regulation: analysis of the X-ray diffraction patterns for
12-residue segment of actin. Biophys J 51: A331. relaxed and contracting muscles. J Mol Biol 75: 35–55.
Haselgrove JC (1972) X-ray evidence for a conformational change in Patchell VB, Gallon CE, Hodgkin MA, Fattoum A, Perry SV and
the actin containing filaments of vertebrate striated muscle. Cold Levine BA (2002) The inhibitory region of troponin I alters the
Spring Harbor Symp Quant Biol 37: 341–352. ability of F-actin to interact with different segments of myosin. Eur
Heeley DH, Golosinska K and Smillie LB (1987) The effects of J Biochem 269: 5088–5100.
troponin T fragments T1 and T2 on the binding of non- Pearlstone JR and Smillie LS (1983) Effects of troponin I plus C on the
polymerisable tropomyosin to F-actin in the presence and absence binding of troponin T and its fragments to a-tropomyosin. J Biol
of troponin I and troponin C. J Biol Chem 262: 9971–9978. Chem 258: 2534–2542.
Huxley HE (1972) Structural changes in the actin- and myosin- Perry SV (1998) Troponin T: genetics, properties and function. J
containing filaments during contraction. Cold Spring Harbor Symp Muscle Res Cell Motil 19: 575–602.
Quant Biol 37: 361–376. Perry SV (2001) Vertebrate tropomyosin: distribution, properties and
Kawamura M and Maruyama K (1970) Electron microscopic particle function. J Muscle Res Cell Motil 22: 5–49.
length of F-actin in vitro. J Biochem 67: 437–457. Perry SV, Cole HA, Head JF and Wilson FJ (1972) Localisation
Kobayashi T, Kobayashi M, Gryczynski Z, Lakowitz R and Collins JH and mode of action of the inhibitory component of the tro-
(2000) Inhibitory region of troponin: Ca2+ dependent structural ponin complex. Cold Spring Harbor Symp Quant Biol 37: 251–262.
and environmental changes in the troponin–tropomyosin complex Phillips GN Jr., Filler JP and Cohen C (1986) Tropomyosin crystal
and in reconstituted thin filaments. Biochemistry 39: 86–91. structure and muscle regulation. J Mol Biol 192: 111–131.
Kojima H, Ishijima A and Yanagida Y (1994) Direct measurement of Resetar AM, Stephens JM and Chalovich JM (2002) Troponin–
stiffness of single actin filaments with and without tropomyosin by tropomyosin: an allosteric switch or steric blocker. Biophys J 83:
in vitro nanomanipulation. Proc Natl Acad Sci USA 91: 12962– 1039–1049.
129. Stein LA, Chock FB and Eisenberg E (1981) Mechanism of the
Kuo-Kang W, Kang B and Rubenstein PA (2000) Tropomyosin- actomyosin ATPase: effect of actin on the ATP hydrolysis step.
dependent filament formation by a polymerization-defective mu- Proc Natl Acad Sci USA 78: 1346–1350.
tant yeast actin (V266G, L267G). J Biol Chem 275: 40594–40600. Syska H, Wilkinson JM and Perry SV (1976) The relationship between
Lehman W, Vibert P, Craig R and Barany M (1996) Actin and the biological activity and primary structure of troponin I of white
structure of smooth muscle thin filaments. In: M. Barany (ed.) skeletal muscle of the rabbit. Biochem J 153: 375–387.
Biochemistry of Smooth Muscle Contraction. (pp. 47–60) Academic Talbot JA and Hodges RS (1981) Synthetic studies on the inhibitory
Press, New York. region of rabbit skeletal troponin I. J Biol Chem 256: 2798–2802.
Lehman W, Rosol M, Tobacman LS and Craig R (2001) Troponin Vibert P, Craig R and Lehman W (1997) Steric-model for activation
organisation on relaxed and activated thin filaments revealed by of muscle thin filaments. J Mol Biol 266: 8–14.
electron microscopy and three dimension reconstruction. J Mol Wegner A and Walsh TP (1981) Interaction of tropomyosin–troponin
Biol 307: 739–744. with actin filaments. Biochemistry 20: 5633–5642.
Lehrer SS, Chai M and Geeves MA (1997) Effect of troponin I (TNI) Xu C, Craig R, Tobacman L, Horowitz R and Lehman W (1999)
on actin S1 ATPase and S1 binding in the absence and presence of Tropomyosin positions in regulated thin filaments revealed by
rabbit skeletal tropomyosin. Biophys J 72: MP 198. cryoelectron microscopy. Biophys J 77: 985–992.