- Email: [email protected]
The regulatory function of the myosin light chains
281
TIBS - December 1976 4 Barton, P.G. (1968) J. Biol. Chem. 243, 38843890 5 Suzuki, Y. and Matsushita, H. (1969) Ind. Health 7, 143- 159 6 Hauser, H.. Darke, A. and Phillips, M.C. (1976) Eur. J. Biochem. 62, 335-344 7 Hauser, H., Kostner, G., Miiller, M. and Skrabal, P. (1976) submitted to Biochemistry 8 Krebs, J., Levine, B.A. and Williams, R. J.P., unpublished results R. 9 Tobias, J.M., Agin, D.P. and Pawlowski, (1962) J. Gen. Physiol. 45, 989-1001 10 Leitch, G. J. and Tobias. J.M. (1964) J. Cell Comp. Physiol. 63,225-232 11 Ohki, S. (1969) Biophys. J. 9, 1195-1205 12 Ohki, S. (1969) J. Colloid Interface Sci. 30, 413420 D. (1970) Adv. 13 Ohki, S. and Papahadjopoulos, Exp. Med. Biol. 7, 155-174 14 Triggle, D. J. (1972) Progr. Surface Membrane Sci. 5,267-331 M.D. 15 Hauser, H., Phillips, M.C. and Barratt, (1975) Biochim. Biophys. Acfa 413, 341-353 D., Vail, W. J., Jacobson, K. 16 Papahadjopoulos, and Poste, G. (1975) Biochim. Biophys. Acta 394, 483491
17 Hauser, H. and Phillips, M.C. (1973) J. Biol. Chem. 248,8585-8591 18 Tocanne, J. F., Ververgaert, P. H. J. Th., Verkleij, A. J. and van Deenen, L. L. M. (1974) Chem. Phys. Lipids 12.201-219 19 Chapman, D., Urbina, J. and Keough, K.M. (1974) J. Biol. Chem. 249,2512-2521 20 Phillips, M.C., Graham, D.E. and Hauser, H. (1975) Nature 254, 154156 21 Hauser, H., Dake, A. and Finer, E.G. submitted to Biochim. Biophys. Acta 22 Rand, R.P. and Sengupta, S. (1972) Biochim. Biophys. Acta 255,484492 23 Goddard, E. D., Kao, 0. and Kung, H.C. (1968) J. Colloid Interface Sci. 26, 616624 24 McLaughlin, S., Bruder, A., Chen, S. and Moser, C. (1975) Biochim. Biophys. Acia 394,304-313 25 Phillips, M.C., Hauser, H. and Paltauf, F. (1972) Chem. Phys. Lipids 8, 127-133 26 Ito, T., Ohnishi, S., Ishinaga, M. and Kito, M. (1975) Biochemistry 14, 30643069 27 Coleman, J.E. and Vallee, B.L. (1961) J. Biol. Chem. 236,22442249
The regulatory function of the myosin light chains John Kendrick-Jones
and Ross Jakes
In molluscan muscles, calcium regulation is mediated by ‘regulatory’ light chains associated with the myosin headrr. This type of ‘regulatory’ light chain appears to be present in all myosins, regardless of whether the myosin contains light chain linked calcium regulation. Although they appear to be ‘structurally’ related, differences in their calcium binding abilities imply that these regulatory light chains may play quite distinct functions in their respective myosins.
OFF in response to these changes in calcium concentration, is due to the presence of specific calcium regulatory proteins associated with these contractile elements. It should be stressed that although muscles may vary in their structure, speed of contraction, tension development and in the location of their calcium control proteins, they appear to be regulated over similar ranges of free calcium ion concentrations. In most vertebrate muscles, the calcium control protein, troponin, is associated with tropomyosin and forms part of the structure of the thin filament (Fig. 1). Troponin is a protein complex consisting of three different subunits, one of which, troponin C, binds calcium with a high affmity. Electron microscopy and X-ray diffraction evidence [2,3] suggests that calcium induced changes in the troponin complex may be transmitted via a movement of tropomyosin, which lies in the groove between the two actin strands, in such a way that it directly affects those MRC Laboratory of’ sites on the actin which are involved in Cambridge CB2 2QH. interaction with the myosin crossbridges.
Ca2+playsavitalroleinavarietyofphysiological processes in eukaryotic cells. Although recent advances have been made in elucidating the role of calcium in many of these systems [1], its involvement in the regulation of muscular contraction is perhaps best understood. It is generally accepted that all muscles contract by a relative sliding of the thick and thin filament past each other, driven by crossbridges projecting from the thick filament which attach to the thin filaments and advance along them in a cyclic and repetitive manner. Contraction is initiated by nervous impulses which by changing the ‘electrical state’ (depolarization) of the muscle membrane and the interconnecting system of transverse tubules within the muscle fibre, ultimately cause the release of calcium from the sarcoplasmic reticulum which surrounds each myofibril. The ability of the myosin crossbridge and thin filament to be switched ON or .7X.-J. and R.J. are at the Molecular Biology. Hills Road, U.K.
Thus, for example, in the absence of calcium, tropomyosin lies out of the groove and directly blocks the myosin attachment sites on the actin monomers whereas in the presence of calcium, tropomyosin moves into the groove thus exposing the sites required for myosin crossbridge interaction. In molluscan muscles, the troponin complex is absent and instead calcium regulation of contractile activity is mediated by specific regulatory-subunits, called ‘regulatory’ light chains, which are associated with each myosin head (Fig. 1) [4]. However, little is known about how these regulatory light chains, under calcium control, effect the transition of the myosin crossbridge from the resting to the active state. The existence of these two distinct calcium regulatory systems poses an interesting fundamental question : What physiological advantage is conferred on a muscle by the possession of either the troponin or myosin-linked regulatory system? Comparative studies on the distribution of these regulatory systems in vertebrate and invertebrate muscles were carried out by Lehman and Szent-Gyorgyi [5]. Using simple functional tests they demonstrated that all vertebrate striated muscles appear to be regulated solely by the troponin-tropomyosin system on the thin filament, myosin-linked regulation whereas appeared to be restricted to the rather more ‘primitive’ invertebrate muscles, such as those of molluscs. Recent evidence, however, indicates that vertebrate smooth muscle may also contain a myosin linked regulatory system [6]. The most interesting observation was that most invertebrate species, for examand ple, annelid worms, nematodes both troponin insects, contain and myosin-linked regulatory systems within the same muscle. These muscles have an obvious physiological advantage since calcium exerts its control on both the components involved in the contractile cycle. Is the absence therefore of troponin linked regulation in molluscan muscles and myosin linked regulation in vertebrate striated muscle due to the lack of expression of their structural genes or to the production of an altered non functional gene product ? In molluscan muscles, small amounts (about 10% of the amount required for function) of troponin-like components are present on the thin filaments [7] and vertebrate striated myosins contain light chains which will ‘apparently’ functionally replace the molluscan calcium regulatory light chain [8]. The presence of only troponin-linked regulation in vertebrate striated muscles and myosin-linked regulation in
282
TIBS
Myosin Filament
) hinge’ region myosin head light chains
Ca 44
‘pawalbumin’
t roponin t ropomyosin actin
da
Thin Filament Fig. I. Diagrammatic representation of myosin filament. with a single crossbridge, and thin filament showing the positions of the calcium binding and regulatory components. A myosin crossbridge is composed of two heads, each containing an ATP hydrolytic and actin binding site. Associated with each head are two types of light chains, one of which, termed ‘regulatory’light chains (solid black) act as calcium regulatory subunits in molluscan muscles and may serve a regulatory role in all myosins. In vertebrate thin jilaments, tropomyosin lies in the groove between the two helical strands of actin monomers and attached to it, at intervals of about 38.5 nm is the calcium regulatory complex, troponin, which consists of three different subunits, one of which, troponin C (solid black) binds calcium with a high affinity. Parvalbumin is a freely soluble calcium binding protein present in the sarcoplasm. It appears to be present in all muscles, but is present in highest concentrations in muscles where the sarcoplasmic reticulum is poorly developed, e.g. fuh muscle, and could serve a role as a modulator of the free calcium ion concentration within the muscle cell [271.
moluscan muscles appears therefore to be a selected adaptation to meet the particular requirements of these muscles. Ancestral ‘muscle’ may have contained both calcium regulatory systems which arose independently and have evolved concurrently in subsequent evolution, but in particular species subsequent modification may have arisen by natural selection. Myosin ‘regulatory’ light chains All the myosins so far studied from a variety of vertebrate and invertebrate muscles have a rather similar subunit structure composed of two classes of light chains associated with the myosin heads (Fig. 1) [8,9]. One class of light chains, with molecular weights in the l&000-21,000 range (in rabbit skeletal myosin referred to as ‘alkali’ light chains [9] may have a structural role, being required to maintain the integrity of the my&in head to ensure correct functional activity, that is, ATP hydrolysis and actin interaction. The second class of light chains (in vertebrate myosins they have a molecular weight of about 19,000, whereas in molluscan myosins about 17,000) appear to be involved in regulating the interaction of the myosin heads with actin and for this
reason have been called ‘regulatory’ light chains [8]. The regulatory function of this class of light chains in molluscan myosins has been established by the selective release of one of the two regulatory light chains in scallop myosin, which results in a complete loss of calcium regulation and partial loss of calcium binding, that is, the myosin is ‘desensitized’ and no longer requires calcium for interaction with actin. The isolated light chain readily recombines with this ‘desensitized’ myosin and both calcium regulation and calcium binding are completely restored [4]. Since scallop myosin is the only calcium regulated myosin from which a regulatory light chain can be reversibly detached without denaturation, it has been used to demonstrate the possible ‘regulatory’ function of this class of light chain in all myosins. ‘Regulatory’ light chains have been prepared from all the myosins examined [8]. They bind tightly to ‘desensitized’ scallop myosin (replacing the released scallop light chain) and if the myosin is complexed with actin, calcium regulation of actinmyosin interaction is completely restored. However, these light chains differ in their ability to restore full calcium binding to desensitized scallop myosin and accord-
- December
1976
ingly can be divided into two groups. In the first group light chains from muscles containing myosin-linked regulation, for examples, molluscan and vertebrate smooth muscles, restore both calcium regulation and calcium binding; they are completely functional. On the other hand in the second group light chains from muscles where the biochemical evidence indicates the absence of myosin-linked regulation, for example, most vertebrate muscles such as fast striated, cardiac and slow muscles, although they restore regulation, have no effect on calcium binding [8]. A possible explanation for the ability of these vertebrate light chains to restore calcium regulation to ‘desensitized’ scallop myosin without calcium binding, is the observation that complete loss of regulation is achieved when only one of the two identical scallop regulatory light chains is released and therefore restoration of calcium regulation may be accomplished by cooperative interactions between the remaining scallop and the added vertebrate light chain. Although there are other highly suggestive indications that this type of calcium regulatory system may require ‘cooperation’ between either the two regulatory light chains or the two myosin heads, there is as yet no direct evidence [8]. The remarkable feature that vertebrate light chains cross react with scallop myosin would imply that ‘regulatory’ light chains and the regions on the myosin head involved in binding these light chains are relatively conservative and have changed little in the millions of years of evolution that separate the molluscs from vertebrates. Calcium binding sites The three-dimensional structure of carp calcium binding parvalbumin established by the X-ray diffraction studies of Kretsinger and Nockolds [lo] which clearly shows two basic structural units, each composed of a calcium binding site in a ‘pocket’ surrounded by helical regions on either side (Fig. 2) has served as a model for identifying potential calcium binding regions in a number of muscle proteins. These crystallographic studies have established that within the calcium binding sites of parvalbumin, calcium is coordinated to oxygen atoms from six amino acids which form the vertices of an octahedron, that is Caz+ 0, (Fig. 2) and within these six coordinating positions, four acidic residues are present. Using these criteria, four calcium binding regions have been identified in troponin C [ 1l] whereas in the rabbit and scallop regulatory light chains only one potential calcium binding site is present [ 12,141. A comparison of the potential calcium
TIBS - December
1976
binding regions in these proteins (Fig. 2) shows striking similarities. Four negatively charged groups (acidic) are present within the six coordinating positions in the sites of troponin C, parvalbumin and scallop light chain and since the binding constant of this site in troponin C is about 1.IO6 M-l [15] it is reasonable to assume that this site in the scallop light chain is the high affinity calcium binding site present in the myosin (Kz 1 - lo6 M-i) [4]. The exact location of the calcium binding sites in scallop myosin had been in doubt since the regulatory light chain once isolated does not bind calcium [4]. The binding site in the rabbit ‘regulatory’ light chain contains only three negatively charged residues within the coordination positions which may explain the lower calcium affinity of this site (K~l*10~ M-l) [16] and the failure of this rabbit light chain to restore the high affinity calcium binding site when it binds to ‘desensitized’ scallop myosin. It might also explain the lack of myosin-linked calcium regulation in vertebrate striated muscles. Given the extensive sequence homology that exists between the parvalbumins, troponin C and the myosin light chains [1 l14,171 it is probable that these proteins evolved from a common ancestral gene by successive gene duplications [ 13,181. However, since they have obviously evolved to perform quite distinct functions within muscle cells and the in vitro evidence indicates they are not interchangeable [19], their overall three-dimensional structures may be quite different. Parvalbumin and troponin C are globular proteins with rather similar structures [20], whereas recent hydrodynamic measurements [2 1] indicate that the isolated molluscan regulatory light chains are extremely asymmetric, with a length ranging from 10 to 14 nm, about the same length as a single myosin head. It remains to be established whether the structure of the light chain in the isolated state is identical to that when it is associated with the myosin head.
283
switching on or off metabolic
pathways
~31.
Although the serine residue has been retained in the primary sequence of the scallop light chain, there is no evidence that molluscan regulatory light chains are phosphorylated [ 141. Phosphorylation may therefore be a more recent evolutionary acquisition and preliminary evidence suggests in vertebrate smooth muscle and platelets that phosphorylation is required for the myosin to be able to interact with
tion available concerning the mechanism of calcium regulation mediated by the light chains one could envisage a simple model where calcium binding to the light chains might change either their relative positions on the myosin heads or the ‘orientation’ of the two heads to facilitate actin binding. The observation that all the myosins so far studied contain ‘regulatory’ light chains which bind to desensitized scallop myosin but differ in their ability to function [8] has provided us with ‘structural analogues’
Phosphorylation
The precise function of the ‘regulatory’ light chains in vertebrate striated and cardiac myosins remains obscure despite indirect evidence from biochemical, hydrodynamic and X-ray diffraction studies indicating some role in controlling the ‘movement’ of the myosin heads [7]. The observation that these regulatory light chains are selectively phosphorylated at a single serine residue located close to the N terminus and calcium binding site, by a calcium requiring light chain kinase [22] further implies a ‘regulatory’ role since phosphorylation as a form of covalent control is a common mechanism for
actin [24,25]. Further clarification of the role of phosphorylation, particularly whether a phosphorylation-dephosphorylation cycle exists in vivo [22] are obviously fruitful areas for further investigation. Mechanism of regulation
Although there is little precise informa-
for investigating the regions of the light chains which are essential for binding to the myosin and those which are responsible for calcium regulation. The recognition of these two groups of ‘regulatory’ light chains implies that molluscan light chains contain a region, which has been retained in all ‘regulatory’ light chains, involved in ‘permanent’ attachment to the
284
TIBS - December
* 12 Troponin
C
c
site
3
4
5
6
7
8
*helix+
912
3
ASP-ala-ASP-gly-GLY-gly-ASP-ile-SEB-oal-lye-GLU
Scallop LC (gUTA)
ASP-val-ASP-arg-ASP-gly-PHE-val-SEX-lye-up-ASP
Rabbit LC W'NB)
ASP-gln-ASN-arg-ASN-gly-ILg-ile-ASP-lye-glu-ASP
rabbit calcium protein
ASP-lye-A8P-1ye-SEB-gly-PHE-I1e-GLU-glu-glu-GLU
binding
Calcium ligating poeitione
x
Y
2
-Y
negative charge6 4
-X
Fig. 2. Diagrammatic representation of the three-dimensional structure of one of the calcium binding sites of carp parvalbumin [9] referred to by Kretsinger and his colleagues as a ‘CD hand’, which has served as a mode2 for recognising the calcium binding regions in other proteins. The calcium binding site is composed of a ‘pocket’ surrounded by helical regions on either side. Calcium is coordinated by oxygen atoms from six amino acids (with their sequence positions in the site region indicated) which lie at the vertices of an octahedron, X, Y and 2. On the left of this figure are the amino acid sequences of the calcium binding sites of troponin C (site 1) [II], scallop ‘regulatory’ light chain [14], rabbit *regulatory’ (DTNB) light chain [IZ] and rabbit calcium binding parvalbumin (site I) [26]. The residues in capitals are the calcium coordination groups which occur at the vertices of the octahedron shown in the diagram, and the number of negative charges in these six positions are indicated.
myosin head and another region whose interaction with the myosin head is altered by calcium. Preliminary indications using selective cleavage with proteolytic enzymes suggests that the C terminal halves of the light chains are involved in this ‘permanent’ association with the myosin head [14]. The corresponding region on the myosin head must be distinct from the actin binding site, since the light chain remains attached during contraction and can be readily released from or combine with the myosin when it is associated with actin [4]. It is probable that multiple electrostatic and hydrophobic interactions are involved in this light chain-myosin association and magnesium probably bound to a specific site on the myosin head is essential, certainly in scallop myosin, for maintaining the head in the correct orientation for light chain attachment. The region of the light chains involved in calcium mediated interaction with the myosin head, by implication, could be the region around the N terminus which contains the calcium binding site [14]. If one speculates that the light chains lie parallel to each myosin head (as in Fig. 1) then, in the absence of calcium, the N terminal region of the light chain in its inhibitory position may directly block the site for actin interaction on the myosin head and contraction is switched Off.
In the presence of calcium, this inhibition is removed as a result of calcium binding which causes a localized charge in conformation of the light chain and allows the myosin head to interact with actin.
I976
Alternatively, in a model where the light chains are associated with the ‘hinge’ connecting the myosin head to the rod (Fig. 1) then calcium binding to the light chain could produce change in this ‘hinge’ region which might influence the orientation of the myosin heads or the ‘actin searching movement of the heads’ observed in X-ray diffraction studies [3]. Obviously we need to understand the mechanism of light chain regulation in greater detail. This will require detailed knowledge of the topography of the myosin head and the positions occupied by the light chains in the different regulated states and also the three-dimensional structure of the light chains to establish the nature of the calcium controlled ‘switch’. References 1 Drabikowski,
W., Strzelecka-Golaszewska, H. and Carafoli, E. (eds) (1974) Calcium Binding Proteins. Proc. Int. Symp. Jablonna July 9-12, 1973,
Elsevier, Amsterdam and PWN-Polish Scientific Publisher 2 Weber, A. and Murray, J. M. (1973) Physiol. Rev. 53, 612-673 3 Huxley, H. E, (1975) in Molecular Basis of Motility: 26th Colloquium Gesellschaft fur Biologische Chemie (Heilmeyer, L., Ruegg, J.C. and
Wieland, T., eds), pp. 9-25, Springer-Verlag, . Berlin 4 Szent-Gyorgyi, A.G., Szentkiralyi, E.M. and Kendrick-Jones, J. (1973) J. Mol. Biol. 74, 179203 5 Lehman, W. and Szent-Gyorgyi, A.G. (1975) J. Gen. Physiol. 66, l-30 6 Bremel, R. D. (1974) Nature 252, 4055407 7 Kendrick-Jones, J. (1975) in Molecular Basis of Motility. 26th Colloquium Gesellschaft fur Biologische Chemie (Heilmeyer, et al., eds), pp. 122-
136, Springer-Verlag, Berlin
8 Kendrick-Jones, J., Szentkiralyi, E. M. and SzentGyorgyi, A. G. (1976) J. Mol. Biol. 104,747-775 9 Lowey, S. and Risby, D. (1971) Nature 234,81-88 10 Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313-3326 11 Collins, J. H., Potter, J. D., Horn, M. J., Wilshire, G. and Jackman, N. (1973) FEBS Lett. 36, 268272 12 Collins, J. H. (1976) Nature 259, 699-700 13 Weeds, A.G. and McLachlan, A. D. (1974) Nature 252,646649 J. and Jakes, R. (1976) in 14 Kendrick-Jones, International Symposium on Myocardial Failure,
Tegerness, Munich, June 1976 (in the press) 15 Potter, J.D. and Gergely, J. (1975) J. Biol. Chem. 250,46284633 16 Morimoto, K. and Harrington, W.F. (1974) J. Mol. Biol. 83, 83-97 17 Pechere, J.-L., Capony, J.-P. and Demaille, J. (1973) Syst. Zool. 22, 533-548
18 Tufty, R. M. and Kretsinger, R.H. (1975) Science 187, 167-169 19 Hitchcock, S. E. and Kendrick-Jones, J. (1975) in Calcium Transport
in Contraction
and Secretion
(Carafoli, E., et al., eds), pp. 447458, North-Holland Publishing Company, Amsterdam 20 Kretsinger, R. H. and Barry, C. D. (1975) Biochim. Biophys. Acta 405, 40-52 21 Stafford, III, W.F. and Szent-Gyorgyi, (1976) Biophys. J. 16, 70a
A.G.
22 Perry, S.V., Cole, H.A., Frearson, N., Moir, A. J.G., Morgan, M. and Pires, E. (1975) in Molecular Basis of Motility. 26th Colloquium Gesellschaft fur Biologische Chemie (Heilmeyer,
et al., eds), pp. 107-121, Springer-Verlag, Berlin 23 Cohen, P. (1976) Trends Biochem. Sci. 1, 3840 24 Adelstein, R. S., Daniel, J.L., Conti, M.A. and Anderson, Jr, W. (1975) Proc. 9th Meeting FEBS 31, 1777186 25 Aksoy, M.O., Williams, D., Sharkey, G.M. and Hartshorne, D. J. (1976) Biochem. Biophys. Res. Commun. 69, 3542 26 Entield, D. L., Ericson, L.H., Blum, H. E., Fischer, E.H. and Neurath, H. (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 130991313 27 Pechere, J.F., Damaille, J., Capony, J.-P.,
Dutruge, E., Baron, G. and Pina, C. (1975) in Calcium Transport
in Contraction
and Secretion
(Carafoli, E., et al., eds), pp. 459-468, North-Holland Publishing Company, Amsterdam
TIBS - December 1976 4 Barton, P.G. (1968) J. Biol. Chem. 243, 38843890 5 Suzuki, Y. and Matsushita, H. (1969) Ind. Health 7, 143- 159 6 Hauser, H.. Darke, A. and Phillips, M.C. (1976) Eur. J. Biochem. 62, 335-344 7 Hauser, H., Kostner, G., Miiller, M. and Skrabal, P. (1976) submitted to Biochemistry 8 Krebs, J., Levine, B.A. and Williams, R. J.P., unpublished results R. 9 Tobias, J.M., Agin, D.P. and Pawlowski, (1962) J. Gen. Physiol. 45, 989-1001 10 Leitch, G. J. and Tobias. J.M. (1964) J. Cell Comp. Physiol. 63,225-232 11 Ohki, S. (1969) Biophys. J. 9, 1195-1205 12 Ohki, S. (1969) J. Colloid Interface Sci. 30, 413420 D. (1970) Adv. 13 Ohki, S. and Papahadjopoulos, Exp. Med. Biol. 7, 155-174 14 Triggle, D. J. (1972) Progr. Surface Membrane Sci. 5,267-331 M.D. 15 Hauser, H., Phillips, M.C. and Barratt, (1975) Biochim. Biophys. Acfa 413, 341-353 D., Vail, W. J., Jacobson, K. 16 Papahadjopoulos, and Poste, G. (1975) Biochim. Biophys. Acta 394, 483491
17 Hauser, H. and Phillips, M.C. (1973) J. Biol. Chem. 248,8585-8591 18 Tocanne, J. F., Ververgaert, P. H. J. Th., Verkleij, A. J. and van Deenen, L. L. M. (1974) Chem. Phys. Lipids 12.201-219 19 Chapman, D., Urbina, J. and Keough, K.M. (1974) J. Biol. Chem. 249,2512-2521 20 Phillips, M.C., Graham, D.E. and Hauser, H. (1975) Nature 254, 154156 21 Hauser, H., Dake, A. and Finer, E.G. submitted to Biochim. Biophys. Acta 22 Rand, R.P. and Sengupta, S. (1972) Biochim. Biophys. Acta 255,484492 23 Goddard, E. D., Kao, 0. and Kung, H.C. (1968) J. Colloid Interface Sci. 26, 616624 24 McLaughlin, S., Bruder, A., Chen, S. and Moser, C. (1975) Biochim. Biophys. Acia 394,304-313 25 Phillips, M.C., Hauser, H. and Paltauf, F. (1972) Chem. Phys. Lipids 8, 127-133 26 Ito, T., Ohnishi, S., Ishinaga, M. and Kito, M. (1975) Biochemistry 14, 30643069 27 Coleman, J.E. and Vallee, B.L. (1961) J. Biol. Chem. 236,22442249
The regulatory function of the myosin light chains John Kendrick-Jones
and Ross Jakes
In molluscan muscles, calcium regulation is mediated by ‘regulatory’ light chains associated with the myosin headrr. This type of ‘regulatory’ light chain appears to be present in all myosins, regardless of whether the myosin contains light chain linked calcium regulation. Although they appear to be ‘structurally’ related, differences in their calcium binding abilities imply that these regulatory light chains may play quite distinct functions in their respective myosins.
OFF in response to these changes in calcium concentration, is due to the presence of specific calcium regulatory proteins associated with these contractile elements. It should be stressed that although muscles may vary in their structure, speed of contraction, tension development and in the location of their calcium control proteins, they appear to be regulated over similar ranges of free calcium ion concentrations. In most vertebrate muscles, the calcium control protein, troponin, is associated with tropomyosin and forms part of the structure of the thin filament (Fig. 1). Troponin is a protein complex consisting of three different subunits, one of which, troponin C, binds calcium with a high affmity. Electron microscopy and X-ray diffraction evidence [2,3] suggests that calcium induced changes in the troponin complex may be transmitted via a movement of tropomyosin, which lies in the groove between the two actin strands, in such a way that it directly affects those MRC Laboratory of’ sites on the actin which are involved in Cambridge CB2 2QH. interaction with the myosin crossbridges.
Ca2+playsavitalroleinavarietyofphysiological processes in eukaryotic cells. Although recent advances have been made in elucidating the role of calcium in many of these systems [1], its involvement in the regulation of muscular contraction is perhaps best understood. It is generally accepted that all muscles contract by a relative sliding of the thick and thin filament past each other, driven by crossbridges projecting from the thick filament which attach to the thin filaments and advance along them in a cyclic and repetitive manner. Contraction is initiated by nervous impulses which by changing the ‘electrical state’ (depolarization) of the muscle membrane and the interconnecting system of transverse tubules within the muscle fibre, ultimately cause the release of calcium from the sarcoplasmic reticulum which surrounds each myofibril. The ability of the myosin crossbridge and thin filament to be switched ON or .7X.-J. and R.J. are at the Molecular Biology. Hills Road, U.K.
Thus, for example, in the absence of calcium, tropomyosin lies out of the groove and directly blocks the myosin attachment sites on the actin monomers whereas in the presence of calcium, tropomyosin moves into the groove thus exposing the sites required for myosin crossbridge interaction. In molluscan muscles, the troponin complex is absent and instead calcium regulation of contractile activity is mediated by specific regulatory-subunits, called ‘regulatory’ light chains, which are associated with each myosin head (Fig. 1) [4]. However, little is known about how these regulatory light chains, under calcium control, effect the transition of the myosin crossbridge from the resting to the active state. The existence of these two distinct calcium regulatory systems poses an interesting fundamental question : What physiological advantage is conferred on a muscle by the possession of either the troponin or myosin-linked regulatory system? Comparative studies on the distribution of these regulatory systems in vertebrate and invertebrate muscles were carried out by Lehman and Szent-Gyorgyi [5]. Using simple functional tests they demonstrated that all vertebrate striated muscles appear to be regulated solely by the troponin-tropomyosin system on the thin filament, myosin-linked regulation whereas appeared to be restricted to the rather more ‘primitive’ invertebrate muscles, such as those of molluscs. Recent evidence, however, indicates that vertebrate smooth muscle may also contain a myosin linked regulatory system [6]. The most interesting observation was that most invertebrate species, for examand ple, annelid worms, nematodes both troponin insects, contain and myosin-linked regulatory systems within the same muscle. These muscles have an obvious physiological advantage since calcium exerts its control on both the components involved in the contractile cycle. Is the absence therefore of troponin linked regulation in molluscan muscles and myosin linked regulation in vertebrate striated muscle due to the lack of expression of their structural genes or to the production of an altered non functional gene product ? In molluscan muscles, small amounts (about 10% of the amount required for function) of troponin-like components are present on the thin filaments [7] and vertebrate striated myosins contain light chains which will ‘apparently’ functionally replace the molluscan calcium regulatory light chain [8]. The presence of only troponin-linked regulation in vertebrate striated muscles and myosin-linked regulation in
282
TIBS
Myosin Filament
) hinge’ region myosin head light chains
Ca 44
‘pawalbumin’
t roponin t ropomyosin actin
da
Thin Filament Fig. I. Diagrammatic representation of myosin filament. with a single crossbridge, and thin filament showing the positions of the calcium binding and regulatory components. A myosin crossbridge is composed of two heads, each containing an ATP hydrolytic and actin binding site. Associated with each head are two types of light chains, one of which, termed ‘regulatory’light chains (solid black) act as calcium regulatory subunits in molluscan muscles and may serve a regulatory role in all myosins. In vertebrate thin jilaments, tropomyosin lies in the groove between the two helical strands of actin monomers and attached to it, at intervals of about 38.5 nm is the calcium regulatory complex, troponin, which consists of three different subunits, one of which, troponin C (solid black) binds calcium with a high affinity. Parvalbumin is a freely soluble calcium binding protein present in the sarcoplasm. It appears to be present in all muscles, but is present in highest concentrations in muscles where the sarcoplasmic reticulum is poorly developed, e.g. fuh muscle, and could serve a role as a modulator of the free calcium ion concentration within the muscle cell [271.
moluscan muscles appears therefore to be a selected adaptation to meet the particular requirements of these muscles. Ancestral ‘muscle’ may have contained both calcium regulatory systems which arose independently and have evolved concurrently in subsequent evolution, but in particular species subsequent modification may have arisen by natural selection. Myosin ‘regulatory’ light chains All the myosins so far studied from a variety of vertebrate and invertebrate muscles have a rather similar subunit structure composed of two classes of light chains associated with the myosin heads (Fig. 1) [8,9]. One class of light chains, with molecular weights in the l&000-21,000 range (in rabbit skeletal myosin referred to as ‘alkali’ light chains [9] may have a structural role, being required to maintain the integrity of the my&in head to ensure correct functional activity, that is, ATP hydrolysis and actin interaction. The second class of light chains (in vertebrate myosins they have a molecular weight of about 19,000, whereas in molluscan myosins about 17,000) appear to be involved in regulating the interaction of the myosin heads with actin and for this
reason have been called ‘regulatory’ light chains [8]. The regulatory function of this class of light chains in molluscan myosins has been established by the selective release of one of the two regulatory light chains in scallop myosin, which results in a complete loss of calcium regulation and partial loss of calcium binding, that is, the myosin is ‘desensitized’ and no longer requires calcium for interaction with actin. The isolated light chain readily recombines with this ‘desensitized’ myosin and both calcium regulation and calcium binding are completely restored [4]. Since scallop myosin is the only calcium regulated myosin from which a regulatory light chain can be reversibly detached without denaturation, it has been used to demonstrate the possible ‘regulatory’ function of this class of light chain in all myosins. ‘Regulatory’ light chains have been prepared from all the myosins examined [8]. They bind tightly to ‘desensitized’ scallop myosin (replacing the released scallop light chain) and if the myosin is complexed with actin, calcium regulation of actinmyosin interaction is completely restored. However, these light chains differ in their ability to restore full calcium binding to desensitized scallop myosin and accord-
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ingly can be divided into two groups. In the first group light chains from muscles containing myosin-linked regulation, for examples, molluscan and vertebrate smooth muscles, restore both calcium regulation and calcium binding; they are completely functional. On the other hand in the second group light chains from muscles where the biochemical evidence indicates the absence of myosin-linked regulation, for example, most vertebrate muscles such as fast striated, cardiac and slow muscles, although they restore regulation, have no effect on calcium binding [8]. A possible explanation for the ability of these vertebrate light chains to restore calcium regulation to ‘desensitized’ scallop myosin without calcium binding, is the observation that complete loss of regulation is achieved when only one of the two identical scallop regulatory light chains is released and therefore restoration of calcium regulation may be accomplished by cooperative interactions between the remaining scallop and the added vertebrate light chain. Although there are other highly suggestive indications that this type of calcium regulatory system may require ‘cooperation’ between either the two regulatory light chains or the two myosin heads, there is as yet no direct evidence [8]. The remarkable feature that vertebrate light chains cross react with scallop myosin would imply that ‘regulatory’ light chains and the regions on the myosin head involved in binding these light chains are relatively conservative and have changed little in the millions of years of evolution that separate the molluscs from vertebrates. Calcium binding sites The three-dimensional structure of carp calcium binding parvalbumin established by the X-ray diffraction studies of Kretsinger and Nockolds [lo] which clearly shows two basic structural units, each composed of a calcium binding site in a ‘pocket’ surrounded by helical regions on either side (Fig. 2) has served as a model for identifying potential calcium binding regions in a number of muscle proteins. These crystallographic studies have established that within the calcium binding sites of parvalbumin, calcium is coordinated to oxygen atoms from six amino acids which form the vertices of an octahedron, that is Caz+ 0, (Fig. 2) and within these six coordinating positions, four acidic residues are present. Using these criteria, four calcium binding regions have been identified in troponin C [ 1l] whereas in the rabbit and scallop regulatory light chains only one potential calcium binding site is present [ 12,141. A comparison of the potential calcium
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binding regions in these proteins (Fig. 2) shows striking similarities. Four negatively charged groups (acidic) are present within the six coordinating positions in the sites of troponin C, parvalbumin and scallop light chain and since the binding constant of this site in troponin C is about 1.IO6 M-l [15] it is reasonable to assume that this site in the scallop light chain is the high affinity calcium binding site present in the myosin (Kz 1 - lo6 M-i) [4]. The exact location of the calcium binding sites in scallop myosin had been in doubt since the regulatory light chain once isolated does not bind calcium [4]. The binding site in the rabbit ‘regulatory’ light chain contains only three negatively charged residues within the coordination positions which may explain the lower calcium affinity of this site (K~l*10~ M-l) [16] and the failure of this rabbit light chain to restore the high affinity calcium binding site when it binds to ‘desensitized’ scallop myosin. It might also explain the lack of myosin-linked calcium regulation in vertebrate striated muscles. Given the extensive sequence homology that exists between the parvalbumins, troponin C and the myosin light chains [1 l14,171 it is probable that these proteins evolved from a common ancestral gene by successive gene duplications [ 13,181. However, since they have obviously evolved to perform quite distinct functions within muscle cells and the in vitro evidence indicates they are not interchangeable [19], their overall three-dimensional structures may be quite different. Parvalbumin and troponin C are globular proteins with rather similar structures [20], whereas recent hydrodynamic measurements [2 1] indicate that the isolated molluscan regulatory light chains are extremely asymmetric, with a length ranging from 10 to 14 nm, about the same length as a single myosin head. It remains to be established whether the structure of the light chain in the isolated state is identical to that when it is associated with the myosin head.
283
switching on or off metabolic
pathways
~31.
Although the serine residue has been retained in the primary sequence of the scallop light chain, there is no evidence that molluscan regulatory light chains are phosphorylated [ 141. Phosphorylation may therefore be a more recent evolutionary acquisition and preliminary evidence suggests in vertebrate smooth muscle and platelets that phosphorylation is required for the myosin to be able to interact with
tion available concerning the mechanism of calcium regulation mediated by the light chains one could envisage a simple model where calcium binding to the light chains might change either their relative positions on the myosin heads or the ‘orientation’ of the two heads to facilitate actin binding. The observation that all the myosins so far studied contain ‘regulatory’ light chains which bind to desensitized scallop myosin but differ in their ability to function [8] has provided us with ‘structural analogues’
Phosphorylation
The precise function of the ‘regulatory’ light chains in vertebrate striated and cardiac myosins remains obscure despite indirect evidence from biochemical, hydrodynamic and X-ray diffraction studies indicating some role in controlling the ‘movement’ of the myosin heads [7]. The observation that these regulatory light chains are selectively phosphorylated at a single serine residue located close to the N terminus and calcium binding site, by a calcium requiring light chain kinase [22] further implies a ‘regulatory’ role since phosphorylation as a form of covalent control is a common mechanism for
actin [24,25]. Further clarification of the role of phosphorylation, particularly whether a phosphorylation-dephosphorylation cycle exists in vivo [22] are obviously fruitful areas for further investigation. Mechanism of regulation
Although there is little precise informa-
for investigating the regions of the light chains which are essential for binding to the myosin and those which are responsible for calcium regulation. The recognition of these two groups of ‘regulatory’ light chains implies that molluscan light chains contain a region, which has been retained in all ‘regulatory’ light chains, involved in ‘permanent’ attachment to the
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* 12 Troponin
C
c
site
3
4
5
6
7
8
*helix+
912
3
ASP-ala-ASP-gly-GLY-gly-ASP-ile-SEB-oal-lye-GLU
Scallop LC (gUTA)
ASP-val-ASP-arg-ASP-gly-PHE-val-SEX-lye-up-ASP
Rabbit LC W'NB)
ASP-gln-ASN-arg-ASN-gly-ILg-ile-ASP-lye-glu-ASP
rabbit calcium protein
ASP-lye-A8P-1ye-SEB-gly-PHE-I1e-GLU-glu-glu-GLU
binding
Calcium ligating poeitione
x
Y
2
-Y
negative charge6 4
-X
Fig. 2. Diagrammatic representation of the three-dimensional structure of one of the calcium binding sites of carp parvalbumin [9] referred to by Kretsinger and his colleagues as a ‘CD hand’, which has served as a mode2 for recognising the calcium binding regions in other proteins. The calcium binding site is composed of a ‘pocket’ surrounded by helical regions on either side. Calcium is coordinated by oxygen atoms from six amino acids (with their sequence positions in the site region indicated) which lie at the vertices of an octahedron, X, Y and 2. On the left of this figure are the amino acid sequences of the calcium binding sites of troponin C (site 1) [II], scallop ‘regulatory’ light chain [14], rabbit *regulatory’ (DTNB) light chain [IZ] and rabbit calcium binding parvalbumin (site I) [26]. The residues in capitals are the calcium coordination groups which occur at the vertices of the octahedron shown in the diagram, and the number of negative charges in these six positions are indicated.
myosin head and another region whose interaction with the myosin head is altered by calcium. Preliminary indications using selective cleavage with proteolytic enzymes suggests that the C terminal halves of the light chains are involved in this ‘permanent’ association with the myosin head [14]. The corresponding region on the myosin head must be distinct from the actin binding site, since the light chain remains attached during contraction and can be readily released from or combine with the myosin when it is associated with actin [4]. It is probable that multiple electrostatic and hydrophobic interactions are involved in this light chain-myosin association and magnesium probably bound to a specific site on the myosin head is essential, certainly in scallop myosin, for maintaining the head in the correct orientation for light chain attachment. The region of the light chains involved in calcium mediated interaction with the myosin head, by implication, could be the region around the N terminus which contains the calcium binding site [14]. If one speculates that the light chains lie parallel to each myosin head (as in Fig. 1) then, in the absence of calcium, the N terminal region of the light chain in its inhibitory position may directly block the site for actin interaction on the myosin head and contraction is switched Off.
In the presence of calcium, this inhibition is removed as a result of calcium binding which causes a localized charge in conformation of the light chain and allows the myosin head to interact with actin.
I976
Alternatively, in a model where the light chains are associated with the ‘hinge’ connecting the myosin head to the rod (Fig. 1) then calcium binding to the light chain could produce change in this ‘hinge’ region which might influence the orientation of the myosin heads or the ‘actin searching movement of the heads’ observed in X-ray diffraction studies [3]. Obviously we need to understand the mechanism of light chain regulation in greater detail. This will require detailed knowledge of the topography of the myosin head and the positions occupied by the light chains in the different regulated states and also the three-dimensional structure of the light chains to establish the nature of the calcium controlled ‘switch’. References 1 Drabikowski,
W., Strzelecka-Golaszewska, H. and Carafoli, E. (eds) (1974) Calcium Binding Proteins. Proc. Int. Symp. Jablonna July 9-12, 1973,
Elsevier, Amsterdam and PWN-Polish Scientific Publisher 2 Weber, A. and Murray, J. M. (1973) Physiol. Rev. 53, 612-673 3 Huxley, H. E, (1975) in Molecular Basis of Motility: 26th Colloquium Gesellschaft fur Biologische Chemie (Heilmeyer, L., Ruegg, J.C. and
Wieland, T., eds), pp. 9-25, Springer-Verlag, . Berlin 4 Szent-Gyorgyi, A.G., Szentkiralyi, E.M. and Kendrick-Jones, J. (1973) J. Mol. Biol. 74, 179203 5 Lehman, W. and Szent-Gyorgyi, A.G. (1975) J. Gen. Physiol. 66, l-30 6 Bremel, R. D. (1974) Nature 252, 4055407 7 Kendrick-Jones, J. (1975) in Molecular Basis of Motility. 26th Colloquium Gesellschaft fur Biologische Chemie (Heilmeyer, et al., eds), pp. 122-
136, Springer-Verlag, Berlin
8 Kendrick-Jones, J., Szentkiralyi, E. M. and SzentGyorgyi, A. G. (1976) J. Mol. Biol. 104,747-775 9 Lowey, S. and Risby, D. (1971) Nature 234,81-88 10 Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313-3326 11 Collins, J. H., Potter, J. D., Horn, M. J., Wilshire, G. and Jackman, N. (1973) FEBS Lett. 36, 268272 12 Collins, J. H. (1976) Nature 259, 699-700 13 Weeds, A.G. and McLachlan, A. D. (1974) Nature 252,646649 J. and Jakes, R. (1976) in 14 Kendrick-Jones, International Symposium on Myocardial Failure,
Tegerness, Munich, June 1976 (in the press) 15 Potter, J.D. and Gergely, J. (1975) J. Biol. Chem. 250,46284633 16 Morimoto, K. and Harrington, W.F. (1974) J. Mol. Biol. 83, 83-97 17 Pechere, J.-L., Capony, J.-P. and Demaille, J. (1973) Syst. Zool. 22, 533-548
18 Tufty, R. M. and Kretsinger, R.H. (1975) Science 187, 167-169 19 Hitchcock, S. E. and Kendrick-Jones, J. (1975) in Calcium Transport
in Contraction
and Secretion
(Carafoli, E., et al., eds), pp. 447458, North-Holland Publishing Company, Amsterdam 20 Kretsinger, R. H. and Barry, C. D. (1975) Biochim. Biophys. Acta 405, 40-52 21 Stafford, III, W.F. and Szent-Gyorgyi, (1976) Biophys. J. 16, 70a
A.G.
22 Perry, S.V., Cole, H.A., Frearson, N., Moir, A. J.G., Morgan, M. and Pires, E. (1975) in Molecular Basis of Motility. 26th Colloquium Gesellschaft fur Biologische Chemie (Heilmeyer,
et al., eds), pp. 107-121, Springer-Verlag, Berlin 23 Cohen, P. (1976) Trends Biochem. Sci. 1, 3840 24 Adelstein, R. S., Daniel, J.L., Conti, M.A. and Anderson, Jr, W. (1975) Proc. 9th Meeting FEBS 31, 1777186 25 Aksoy, M.O., Williams, D., Sharkey, G.M. and Hartshorne, D. J. (1976) Biochem. Biophys. Res. Commun. 69, 3542 26 Entield, D. L., Ericson, L.H., Blum, H. E., Fischer, E.H. and Neurath, H. (1975) Proc. Nat. Acad. Sci. U.S.A. 72, 130991313 27 Pechere, J.F., Damaille, J., Capony, J.-P.,
Dutruge, E., Baron, G. and Pina, C. (1975) in Calcium Transport
in Contraction
and Secretion
(Carafoli, E., et al., eds), pp. 459-468, North-Holland Publishing Company, Amsterdam