protein of M r 97 K has recently been cloned and sequenced (Lanar, Pearce, ..... expert technical assistance, and Louise Seidel for help with this manuscript.
Bioscience Reports, Vol. 7, No. 1, 1987
Amino Acid Sequence and Structural Repeats in Schistosome Paramyosin Match Those, of Myosin Carolyn Cohen, 1 David E. Lanar 2 and David A. D. Parry 3 Received February 2, 1987 KEY WORDS: amino acid sequence; paramyosin; schistosome.
The cDNA encoding about half of an antigenic non-surface schistosome parasite protein of M r 97 K has recently been cloned and sequenced (Lanar, Pearce, James and Sher (1986) Science 234:593 596). Analysis of this sequence, together with the properties of the native protein, reveals that this protein is paramyosin, the hitherto unsequenced core protein of myosin filaments in invertebrate muscle. In this report we analyze in more detail the partial amino acid sequence of schistosome paramyosin and describe electron microscope studies of the native protein and its aggregates. We show a close correspondence between the structures of paramyosin and the myosin rod that is required for these proteins to assemble together in muscle thick filaments.
INTRODUCTION The partial amino acid sequence of schistosome paramyosin, and the overall amino acid composition of the native protein, confirm earlier findings that the structure of the molecule is an c~-helical coiled coil (Cohen and Szent-GyiSrgi, 1957; Cohen and Holmes, 1963). As in other paramyosins, there are no proline residues. The sequence displays through the 7-residue (heptad) repeat (a-b-c-d-e-f-9),, in which a and d are generally apolar, that is characteristic of many e-helical coiled-coil proteins (Crick, 1953; McLachlan and Stewart, 1975) (Fig. 1). This heptad pattern is less regular than that of tropomyosin in containing fewer apolar residues in a (Hodges et al., 1972; t Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02254. 2 Immunology and Cell Biology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892. 3 Department of Physics and Biophysics, Masgey University, Palmerston North, New Zealand. 11 0144-8463/87/0100-0011505.00/0 9 1987 Plenum Publishing Corporation
12
Cohen, Lanar and Parry
McLachlan and Stewart, 1976), but is strikingly similar to that of the rod of myosin (McLachlan and Karn, 1983; McLachlan, 1984), including the overall distribution of charged groups (see below). Application of the Robson (Garnier et al., 1978) or ChouFasman (1974) techniques indicates that there are a few short regions not predicted to be e-helical; these are likely to adopt an e-helical conformation, however, through the influence of bordering sequences (cf. e-tropomyosin). The coiled coil is stabilized by two types of interaction: non-polar and ionic. Nonpolar residues at positions a and d form a "core" of interlocking hydrophobic side chains. Charged groups, particularly those in positions e and g, can form intramolecular salt bridges (McLachlan and Stewart, 1975; Parry et al., 1977). The number of these possible interactions depends on the orientation and register of the two e-helices. For an unstaggered structure the score for paramyosin (+26) corresponds to 0.41 ionic interactions per heptad pair (cf. nematode myosin (+ 38) 0.24, and tropomyosin (+ 30) 0.74). As in all other two-chain e-fibrous proteins, a parallel in register arrangement of chains is preferred for paramyosin.
RESULTS Homology searches and Fourier analysis of the amino acid sequence of the paramyosin reveal additional regularities related to a 28-residue repeat. The dominant periods in acidic (D, E), basic (K, R) and apolar residues (L, V, I, M, F, Y, A) are 9.39 (28/3), 9.39 (28/3) and 3.50 (28/8) residues respectively. The acidic and basic periods are almost out of phase with one another, indicating that each 28-residue segment can be subdivided into zones of alternating positive and negative charge. These periodicities are similar to those in myosin rod from various sources (Parry, 1981; McLachlan and Karn, 1983; McLachlan, 1984; Strehler et al., 1986). In the nematode myosin rod, McLachlan and Karn (1982) showed that in four places an extra "skip" residue occurred after position c in the heptad repeat, and that this resulted in interruptions in the 28-residue repeat pattern. In schistosome paramyosin, similar skip residues (196 and 422) have been found in positions analogous to those in both nematode and rat myosin. An extra skip (225), however, is found for paramyosin (Fig. 1). On the basis of homology, we would infer that this skip residue is also present in myosin, but that a new deletion, previously unrecognized, is present in the myosin sequence. In both paramyosin and the myosin rod, there are thus two skip residues separated by only 28 residues, whereas all other adjacent skips are separated by 7 • 28 residues. The effect of the deletion in this region of the myosin rod, together with the proximity of the skip residues, points to a conserved feature of the coiled coil which may have some special structural significance. Self-homology searches that exclude the skip residues reveal longer repeats in the paramyosin sequence of 196 residues (7 x 28) and 252 residues (9 • 28). The 196residue period corresponds to that seen in both nematode and rat myosin (McLachlan and Karn, 1983; Strehler et al., 1986). The 252-residue period is not found in the myosin rod and there are insufficient data as yet to show whether this homology is present in the entire paramyosin sequence. On the basis of the amino acid repeats, there is strong evidence that paramyosin as well as the myosin rod has evolved from a
Paramyosin Amino Acid Sequence
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Fig. l. Comparisonof partial amino acid sequencesof (a) schistosomeparamyosin, (b) nematodemyosin rod (McLachlanand Karn, 1983)and (c) rat myosinrod (Strehleret al., 1986)in a regionof homology.The amino acid sequencenumbers for nematode myosin refer to the rod portion only (McLachlanand Karn, 1983). Apolar residues in positions a and d permit the adoption of a coiled-coilstructure. Note that to maximize homology,an additional skip residue (E 773/E 1610)has been assigned in the myosinsequences, but that an additional deletion [x] is now required. The charged and apolar residues are very highly conserved between the paramyosin and myosin sequences.
28-residue ancestral peptide by a series of gene duplications (McLachlan, 1984). Recent studies of the genomic sequence of mammalian myosin heavy chains suggest, however, that other more complex mechanisms may have produced these simple structural repeats (Strehler et al., 1986). The same regularity in the disposition of the acidic and basic residues in both paramyosin and myosin indicates that the molecular assembly of both proteins will be largely specified by intermolecular ionic interactions. These may be estimated by treating individual molecules as linear arrays of charged and uncharged amino acids (Hulmes et al., 1973). Maxima in the interaction curves for paramyosin occur at relative axial staggers of 28(m +0.5) residues, as found for myosin (Parry, 1981; McLachlan and Karn, 1982). Assuming an axial rise per residue of 1.485 •, these staggers correspond to distances of 4.16 (m + 0.5) A. For example, ifm = 3, the stagger is 145.6 A; for m = 10, the stagger is 436.8 A; both values are found in paramyosin and myosin assemblies (see below). Note that the common periodicities in the acidic and the basic residues are identical in both paramyosin and myosin. This finding strongly implies that these molecules are designed to assemble together. The periodicity of 196 residues leads to enhanced maxima in the ionic interaction curve at relative axial staggers of 291 (n + 0.5) A. Thus the 145 A periodicity in the thick filament is jointly specified by the conditions m = 3 and n = 0, and 435 A periodicity by m = 10 and n = 1. The 725 A period characteristic of both the paramyosin core of invertebrate thick filaments and paramyosin paracrystals found in vitro would arise from m = 17 and n = 2. The shorter 290 A (2/5 x 725 A) "shift" between "subfilaments" found in paramyosin filaments in vitro (Cohen et al., 1971) is the basis of the so-called "Bear-Selby" net characterizing the paramyosin core of native thick filaments. This would result from a combination of molecular shifts of 725 A and 435 A (cf. McLachlan and Karn, 1982). The presence of regularities in the linear disposition of the acidic and basic residues in both paramyosin and myosin also indicates that both parallel and
14
Cohen, Lanarand Parry
antiparalM modes of interaction are possible. Since the thick filament in invertebrate muscles is a bipolar arrangement of myosin molecules arrayed on a bipolar paramyosin core (Cohen et al., 1971; Szent-GyiSrgi et al., 1971), it is likely that the antiparallel alignment of molecules at the center of the thick filament is also specified by ionic bridges or by the linking of aligned clusters of acidic residues via divalent cations (cf. Parry, 1975, 1981). The paramyosin sequence displays other distinctive features. Paramyosin is unusual in its high arginine/lysine ratio. The arginine content of paramyosin is in fact greater than or equal to that in myosin in all positions of the heptad. The arginines are randomly distributed in paramyosin except for position d where only one arginine is found. Note that this arginine is conserved also in myosin and may signify a weak spot in coiled-coil structure (Parry, 1982). Paramyosin also contains a higher percentage of apolar residues and a lower percentage of charged residues in b, c and f than does myosin, features consistent with a more internal role for paramyosin than for myosin. This feature in the sequence, together with the number of intramolecular ionic interactions, suggests that paramyosin is more "stable" than the myosin rod. Apolar residues, in addition to ionic ones, may also be significant in specifying the aggregation of paramyosin with myosin and with itself. We have also examined aggregates produced from purified schistosome paramyosin in order to demonstrate the relationships between the repeats in the amino acid sequence and those in the structures formed by this protein. Rotary shadowed preparations (Fig. 2a) show that the molecules are about 1210 + 50 A in length and are relatively straight, although occasional bends near an end are observed. This length, which agrees with the 97 K subunit weight from SDS gels (Lanar et al., 1986), can be determined more precisely by examination of paracrystals formed in vitro. Paracrystals with a characteristic 145 A repeat (Fig. 2b) and a 725 A repeat (Fig. 2c) have been obtained. The band pattern in the latter can be accounted for by oppositely directed arrays of molecules 1220_+20 A in length that overlap with shifts of both 725 A and 435 A, as shown in Fig. 2c (cf. Cohen et al., 1971). Similar paracrystalline forms have been observed with paramyosin from many molluscs (Cohen et al., 1971), nematodes (Waterston et al., 1974) and a wide variety of other invertebrates (Weisel, 1975; Winkelman, 1976). Although the paracrystals from different paramyosins display distinctive small differences, the large-scale periodicities are the same. As in the case of myosin, highly conserved structural features are characteristic of all paramyosin molecules.
DISCUSSION The close correspondence shown here between the periodicities of paramyosin and the rod region of myosin is required for these proteins to assemble together in the thick filaments of invertebrate muscle. The solution to the detailed packing of the molecules in the filaments will require knowledge of the coiled-coil pitch length of both paramyosin and myosin molecules, the role of the skip residues (and related deletions), and modes of packing for e-helical coiled-coils. Although the paramyosin provides a stable scaffolding upon which the myosin molecules assemble, slight alterations in the
Paramyosin Amino Acid Sequence
Fig. 2. Electron microscopy of schistosome paramyosin. The protein was purified by a modification of the procedure of Harris and Epstein (1977). (a) Rotary shadowed paramyosin. The molecules are about 1210 + 50 A (standard deviation) long and 20 A wide and are fairly straight compared with the rod region of myosin (cf. Flicker et al., 1983). Replicas were prepared by rotary shadowing with platinum (Flicker et al., 1983). Measured dimensions may vary + 20 A, depending on the thickness of the platinum deposit. Scale bar, 1000 •. (b) Paramyosin paracrystal showing a 145 A repeat. The period (144 + 5 A) is divided into three bands. The protein was dissolved (1.5 mg/ml) in 1 M KCI, 0.05 M Tris, pH 8, 0.001 M Dithiothreitol (DTT), then dialyzed against 0.05 M MgC12, 0.05 M Tris (pH 8), 0.001 M DTT. Scale bar, 725 A. (c) Paramyosin paracrystal showing a 725/~ repeat. The protein was dissolved as in (b) but with 0.05 M KSCN present, then precipitated with 0.05 M BaClz. The period of the band pattern is 735 + 20 A. The band pattern is bipolar, showing dihedral symmetry, and is divided into a broad ( ~ 170 ~) and narrow (72 A) stain-excluding zone, together with a dark.staining zone bisected by the latter. The band pattern (so called DII form) can be accounted for by oppositely directed arrays of molecules which do not have end-to-end bonding. Intermolecular staggers include both 725 A and 435 A shifts (cf. Cohen et al.,, 1971). Paracrystals in (b) and (c) were negatively stained with 1 ~ uranyl acetate. Scale,. bar, 725 A.
15
16
Cohen, Lanar and Parry
charge profile of either molecule (through, for example, phosphorylation) might be expected to alter dramatically the local interactions between paramyosin and myosin. Such changes might have functional significance for specialized contractile states such as "catch" (Cohen, 1982; Castellani and Cohen, 1987). ACKNOWLEDGEMENTS We t h a n k D r Alan Sher for support and encouragement, Carla Abramcheck for expert technical assistance, and Louise Seidel for help with this manuscript. This work was supported by grants from N I H (AM17346) and the Muscular D y s t r o p h y Association (to CC), and from N S F (DMB85-02233) (to C C and Peter Vibert); and grants to Dr A. Sher from the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and Training in Tropical Diseases and the E d n a McConnell Clark F o u n d a t i o n (DEL).
REFERENCES Castellani, L. and Cohen, C. (1987). Science 235:334-337. Chou, P. Y. and Fasman, G. D. (1974). Biochemistry 13:22~245. Cohen, C. (1982). Proc. Natl. Aead. Sci. USA 79:3176-3178. Cohen, C. and Holmes, K. C. (1963). J. Mol. Biol. 6:423M32. Cohen, C. and Szent-Gy~Srgyi,A. G. (1957). J. Am. Chem. Soc. 79:248. Cohen, C., Szent-Gy6rgyi, A. G. and Kendrick-Jones, J. (1971). J. Mol. Biol. 56:223 237. Crick, F. H. C. (1953). Acta Cryst. 6:689-697. Flicker, P. F., Wallimann, T. and Vibert, P. (1983). J. Mol. Biol. 169:723-741. Gamier, J., Osguthorpe, D. J. and Robson, B. (1978). J. Mol. Biol. 120:97-120. Harris, H. E. and Epstein, H. F. (1977). Cell 10:709-719. Hodges, R. S., Sodek, J., Smillie, L. B. and Jurasek, L. (1972). Cold Spring Harbor Syrup. Quant. Biol. 37:299-310. Hulmes, D. J. S., Miller, A., Parry, D. A. D., Piez, K. A. and Woodhead-Galloway, J. (1973). J. Mol. Biol. 79:137-148. Lanar, D. E., Pearce, E. J., James, S. L. and Sher, A. (1986). Science 234:593-596. McLachlan, A. D. (1984). Ann. Rev. Biophys. Bioeng. 13:167-189. McLachlan, A. D. and Karn, J. (1982). Nature 299:226-231. McLachlan, A. D. and Karn, J. (1983). J. Mol. Biol. 164:605.626. McLachlan, A. D. and Stewart, M. (1975). J. Mol. Biol. 98:293-304. McLachlan, A. D. and Stewart, M. (1976). J. Mol. Biol. 103:271-298. Parry, D. A. D. (1975). J. Mol. Biol. 98:519-535. Parry, D. A. D. (1981). J. Mol. Biol. 153:459-464. Parry, D. A. D. (1982)~ Biosci. Rep. 2:1017-1026. Parry, D. A. D., Crewther, W. G., Fraser, R. D, B. and MacRae, T. P. (1977). J. Mol. Biol. 113:449-454. Strehler, E. E., Strehler-Page, M. A., Perriard, J. C., Periasamy, M. and Nadal-Ginard, B. (1986). J. Mol. Biol. 190:291-317. Szent-GyiSrgyi, A. G., Cohen, C. and Kendrick-Jones, J. (1971). J. Mol. Biol. 56:239-258. Waterston, R. H., Epstein, H. F. and Brenner, S. (1974). J. Mol. Biol. 90:285-290. Weisel, J. (1975). J. Mol. Biol. 98:675-681. Winkelman, L. (1976). Comp. Biochem. Physiol. 55B:391-397. N o t e A d d e d in P r o o f
After this ms. was submitted, we learned that similar features have been discovered in the partial amino acid sequence of nematode paramyosin deduced from D N A sequence studies of the cloned uric-15 gene of C a e n o r h a b d i t i s e l e g a n s (H. K a g a w a , J. Karn, and A. D. McLachlan, private communication).
