Download Amino acid sequence and structural repeats in schistosome

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.
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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).
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Chou, P. Y. and Fasman, G. D. (1974). Biochemistry 13:22~245.
Cohen, C. (1982). Proc. Natl. Aead. Sci. USA 79:3176-3178.
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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.
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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).