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Acetylcholinesterase: The structure of crystals of a globular form from electric eel
J. Mol. Biol. (1975) 97, 55-60
Acetylcholinesterase: The Structure o f Crystals o f a Globular F o r m from Electric Eel Cr~us C~oTm,t Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England AND
WALO LEUZ~GER
Instituto de Biofisica, Universidade ]Federal Do Rio de Janeiro, Brazil (Received 30 January 1975, and in revised form 2 May 1975) Acetylcholinesterase prepared from electric eel by the method of Leuzinger & Baker (1967) has a sedimentation coefficient of 11 S and has been described as a "globular" form of the enzyme. Here we report the crystallization of this form and its examination by sodium dodecyl sulphate acrylamide gel eleetrophoresis. The crystals belong to space group P6222 (or P6422) and have the unit cell dimensions a = b ~- 187 A, c = 292 A, y = 120°. The crystal structure and acrylamide gel pattern indicate that the molecule is a tetramer with maximum essential dimensions 93 A × 81 A × 97 ~_. The tail stmmture fmmd in the native (9 S, 14 S and 18 S) forms of the enzyme may be formed by a long s-helix from each subunit. 1. I n t r o d u c t i o n
Nerve impulses are transmitted at cholinergic synapses b y the release of acetylcholine from one cell and its diffusion to a second cell where its interaction with receptors causes depolarization of the potential across the membrane. Also associated with the post-synaptic membrane is acetylcholinesterase which hydrolyses acetylcholine to choline and acetic acid and so terminates the depolarizing action. Acetylcholinesterase (EC 3.1.1.7) has been purified from the electric tissue of Electrophorus electricus b y a variety of methods, most of which initially involve homogenizing the tissue and then storing it in toluene for long periods (Lawler, 1961 ; Kremzner & Wilson, 1963; Leuzinger & Baker, 1967; Massouli~ & Rieger, 1969; Dudai et ed., 1972a) or treating it with trypsin (Dudai et al., 1972a). These methods give five forms of the enzyme in varying proportion (Grafius & Millar, 1965; Massouli6 & Rieger, 1969; Massouli6 eta/., 1970,1971; Dudai e$ a/., 1972a). The five forms can be denoted b y their approximate sedimentation coefficients: three are "native", 18 S, 14 S and 9 S, and two are "globular", 11 S and 8 S. The native forms are distinguished b y their ability to aggregate at low ionic strength (Grafius & MiUar, 1965 ;-Massouli6 & Rieger, 1969; Dudai et al., 1972a) and b y their asymmetric structure (Massouli6 et al., 1971). Present address, to which inquiries concerning this paper should be sent: Service de Bioehimie Cellulaire, Institut Pasteur, 28 rue de Docteur Roux, 75015 Paris, France. 55
56
C. C H O T H I A
A N D W. L E U Z I N G E R
Dudai and Ms colleagues (1972b) purified acetylcholinesterase from electric eel by homogenizing the electric organ and directly passing the supernatant over an af~nlty column, With this method of preparation, only the native 18 S and 14 S forms are obtained in significant quantities. Native forms are converted to globular forms by the action of trypsin (Dudai & Silman, 1971), sonication (Massouli4 et al., 1971) or by allowing solutions to age. This last method involves proteolysis by endogeneous proteases (Rieger et al., 1972). There is good agreement among the values reported for the molecular weight of the 11 S form: 240,000 (Lawler, 1961), 230,000 (Kremzner & Wilson, 1963), 260,000 (Leuzinger et al., 1969), 224,000 (Froede & Wilson, 1970), 240,000 (Pavlic & Wilson, 1970), 260,000 (Miliar & Grafins, 1970); though a value of 320,000 has also been reported (I)udai et al., 1973). The molecular weight of the 18 S form is reported to be 1,100,000 (Bonet a/., 1973; Dudai e~ al., 1973); and the 14 S form to be 780,000 (Bonet al., 1973) and 750,000±150,000 (Dudai et al., 1973). Electron micrographs of the 11 S forms have been published by Changeux et al. (1969), of all five forms by Rieger et a/. (1973) and of the 18 S, 14 S and 11 S forms by I)udai et al. (1973). These workers come to similar conclusions. The 11 S form is seen as a globular particle --~110/~ in diameter with a tetramerie structure formed by subunits each about 50 A in diameter. Occasionally trimers and dimers are seen and are thought to arise from the degradation of tetramers. The native forms however consist of a "head" composed of the 50 A globular subunits and a long semi-rigid "tail". The 9 S molecule has three to four subunits in the head, the tail is ~-~20 A wide and the molecule ~-~500 A long. The head of the 14 S molecule has six to eight subunits and the head of the 18 S molecule at least ten subunits. Both the 14 S and 18 S particles axe about 500 ~ long, and the tails 20 to 30 ~ wide. Rieger et al. (1973) occasionally find short tails, ~100/~, attached to the globular forms. Acetylcholinesterase purified by the method of Leuzinger & Baker (1967) was used in the experiments described here. This form of the enzyme is "globular" (Massouli~ et al., 1971, and see below), has a molecular weight of 260,000 and four subunits (Leuzinger et al., 1969); and a sedimentation coefficient of 11.1 S (Massouli~ et al., 1970). We report here the crystal and subunit structure and molecular dimensions of this form and we discuss the relation of the results to those obtained by other workers who used different techniques.
2. Crystallization and Preliminary X-Ray Data The acetycholinesterase used here had a specific activity of 750 mmol acetylcholine hydrolysed per hour per mg protein. Crystals were grown by the dialysis of a protein solution against solutions of ammonium sulphate. The protein (10 rag/rot in 0-05 •-phosphate buffer, pH 7-4) was placed in small plastic containers which held 5 to 25 pl of solution and had one end open. The open end of the containers was closed with "Visking" dialysis tubing (Scientific Instrument Center Ltd, London) and they were placed in various buffered salt solutions. Salt solutions with 37 to 42~o saturated ammonium sulphate and 0.05 M-phosphate buffer (pH 7.4) gave the best crystals that we obtained. Crystals grew as hexagonal prisms with trigonal termination and are similar in appearance to those obtained by Leuzinger et ed. (1968). Small crystals, 10 to 60 pm long, grew in a few weeks at room temperature and at 4°C. After this, all crystallization vessels were transferred to a cold room (4°C).
ON CRYSTALS OF ACETYLCHOLINESTERASE
57
Several crystals ~100/~m long, grew after a few months and 3, 200 to 300/~n long, after 3 years. X-ray diffraction photographs of the crystals were taken at 4°C on a Nonius Precession camera with an Elliot rotating-anode X-ray tube. At this temperature, crystals in the X-ray beam diffract for about 2 days; at room temperature the diffraction pattern dies very quickly. On "still" photographs, reflections can be seen to a resolution of at least 3.9 A. Plate I shows 2 ° precession photographs of (a) the hk0 plane of the reciprocal lattice and (b) the 0/d plane. The unit cell dimensions are a -~ b ~- 187 A; c ~ 292 A and y ~ 120°. Taken together, the 2 photographs indicate the Laue symmetry of the crystal to be 6]mmol. In the (Okl) zone, the only 001 reflections present are 003, 006 and 009. These results, together with the necessity for an enantiomorphie space group, imply that the space group of the crystals is P6222 (or P6~22). The molecular weight of this form of acetylcholinesterase is 260,000 (Leuzinger et al., 1969). The space group P6222 has 12 general positions. If we assume one molecule per asymmetric unit, the ratio of the volume of the unit cell to the molecular weight of protein in the cell, V~, is 2.6, a common value for protein crystals (Matthews, 1973). Two molecules in the asymmetric unit give a VM value of 1-3 which is unreasonably low and half a molecule in the asymmetric unit gives a V~ value of 5-2 which is unlikely, especially in view of how well the crystals diffract X-rays (Nlatthews, 1973). Thus, it is very likely that these crystals have one molecule in the asymmetric unit. This implies that 53 ~/o of the crystal volume is solvent. In the 0/d diffraction pattern those lines for which l ~- 3n are of greater intensity than those for which 1 ~ 3n. Similarly, on the "still" photographs the rings due to the h~l, hb2 and h/c4 layers are of particularly low intensity. Thus at low resolution the c axis has a pseudo-repeat of c/3 (97/~). The relevance of this point to the shape of the molecule is discussed below.
3. Examination by Acrylamide Gel Electrophoresis The acetylcholinesterase preparation from which the crystals were grown was examined by disc gel electrophoresis following the procedure of Davis (1964). The gels were made of 7.5~/o aerylamide and stained for protein with Coomassie blue and for acetyleholinesterase activity by the direct colouring thiocholine method of Karnovsky & Roots (1964). Electrophoresis of the protein in Tris]glycine buffer, pH 8.3, showed only one component. This component was stained by the direct colouring thiocholine method when acetylthiocholine was used, but not with butylthiocholine, indicating that the enzyme is an acetylcholine specific cholinesterase (Plate II(a)). The protein band was cut out of the gel and, following the procedure of Weber & Osborn (1969), was re-run on a 5~/o acrylamide gel in the presence of sodium dodecyl sulphate and fl-mercaptoethanol, to give the pattern shown in Plate II(b). The same pattern was obtained using the initial protein preparation and carboxymethylated protein. There are two main components: the major has a molecular weight ~58,000 and the minor a molecular weight ~27,000, and traces of other components. The band for the minor component is fairly broad and may be split which indicates some heterogeneity. The molecular weight and subunit structure of this preparation of acetylcholinesterase were previously examined by Leuzinger et al. (1969) using sedimentation equilibrium. They found the molecule to have a molecular weight of 260,000 and to
58
C. C H O T H I A A N D W. L E U Z I N G E R
be c o m p o s e d o f f o u r s u b u n i t s e a c h o f 64,0004-4000. T h e r e was n o t r a c e o f t h e 27,000 m o l e c u l a r w e i g h t p e p t i d e f o u n d h e r e b u t t h e s e a u t h o r s n o t e d t h a t in a p r e l i m i n a r y sedimentation velocity run approximately 15% of the optical density trailed behind t h e m a j o r b o u n d a r y a n d t h a t this f r a c t i o n was r e m o v e d before t h e m o l e c u l a r w e i g h t s were d e t e r m i n e d .
TABLE 1
Molecular weights of the s~b~nits in globular acetylcholinesterase Molecular weight 11 S form 260,000 224,000
8 S form ~ > ~
> 102,000 134,000
1" 320,000
Reference
......
>
-> 160,000
> ~ ÷
t
Subunits 64,0005 49,000 65,000
Leuzinger et al. (19601 Froede & Wilson (1970) Millar et al. (19731
60,000 75,000 64,000§ 88,000
Berman (19731 Dudai & Silman (1971) Dudai et al. (1972a,1073)
70,000][ 58,000¶
Roscnberry et al. (19741 This work
j" Not determined. ~t Same preparation as used here (see text). § In a more recent publication these authors note that their preparations contain peptides of 25,000. II These authors find peptides with a molecular weight of 2%000 and 23,500 (see text). ¶ Peptides of N 27,000 molecular weight also present (see text).
t
c/3 !
J..~3 a 4
FIG. 1. The asymmetric unit of P6~22 or P6422 can be seen as a box a/2, ~/3a/4, c]3 surrounded by 8 2-fold axes. The box drawn here has its centre at z --~ 0. An alternative and equivalent set of boxes can be drawn with their eentres at z = 1/6.
PLATE L 2 ° sereenless precession photographs of eryst~als of acetyleholinesterase. (a) The hk0 reciprocal lattice a n d (b) the O]cZ reciprocal lattice.
[ f ~ i ~ p. 5s
(b) PLATE I•. Aerylamide gel electrophoresis of globular acotyleholinesterase. (a) In Tris/glyeine buffer (pH 8.3) and (b) in sodimn dodecyl sulphate and ~-mercaptoethanoh
ON CRYSTALS OF ACETYLCHOLINESTERASE
59
The subunit-structure of other preparations of 11 S acetylcholinesterase have been investigated by several groups. Their results are summarized in Table 1 and fall into two classes: either molecules have four subunits of molecular weight ,-~60,000 or two of ~60,000 and two of 75,000 to 88,000. (The number of active sites on the 11 S molecule has been reported as 4 (Kremzner & Wilson, 1963; Froede & Wilson, 1970; Rosenberry & Bernhard, 1971; Mooser et al., 1972; Berman, 1973), 3.8 (Chen e~ al., 1974), ~-~3 (Dudai et al., 1972a) and 2 (Leuzinger, 1971).) The heterogeneity of preparations of globular acetylcholinesterase has been examined in detail by Rosenberry and his colleagues (Chen et al., 1974; Rosenberry e$ al., 1974) who conclude that it arises from differing degrees of the proteolytic action involved in converting the native forms of the molecule to the globular forms, Two types of proteolysis occur in the conversion. The first type results in the loss of the tail structure found in the native forms of the enzyme. The second type cleaves the subunits at one or two sites but release of measurable peptides only occurs when the molecules' disulphide bridges are reduced. This second type of proteolysis occurs to a partial and variable extent and produces peptides of 27,000 and 23,500 molecular weight: that is, peptides whose molecular weight is the same as that of the minor component observed here. Inspection of the published sodium dodecyl sulphate acrylamide gel patterns of the native forms of the enzyme (Dudai et al., 1972b, Powell et al., 1973) indicates that they only contain subunits of molecular weight ~-~85,000.
4. Discussion I t would be interesting to know whether the structure seen in the electron micrographs can be related to the crystallographic results. The asymmetric unit of the space group P6222 can be seen as a box, dimensions a[2, ~¢/3a]4, 0]3, surrounded by eight 2-fold symmetry axes (Fig. 1). With the unit cell a ~ b ~ 187 A, c ~ 292 A, this box has the dimensions 93 A × 81 A × 97 A. In the a,b plane the 2-fold symmetry axes which occur at ~ ~ 0, relate adjacent boxes which form a square layer structure. The 62 (or 64) screw axis stacks three of these layers on top of each other to give the unit cell. In this crystal no molecular symmetry coincides with the crystallographic symmetry. Therefore, the molecule cannot cross a 2.fold axis, so that, if the molecule has the globular shape seen in electron micrographs, it must essentially sit inside the asymmetric unit box. This implies that the crystal will have a layer structure and, at low resolution, a pseudo c spacing of c/3. As noted above, the X-ray diffraction pattern at low resolution does have a pseudo spacing of c]3, cleaTly indicating that the crystal has a layer structure. Thus, the crystallographic evidence suggests that this large molecule has a globular shape with the maximum essential dimensions of 93 -~ × 81 A × 97 A. This is a little smaller than the molecule seen in the electron microscope which is ~-~110 A in diameter. This discrepancy is probably due to some flattening of the molecule in the electron microscope (Finch et al., 1974). As noted above, the conversion of the native forms of the enzyme to the globular forms involves the partial or total removal of a ,~ 25,000 molecular weight peptide from each subunit and the concomitant disappearance of the tail structure. I f this peptide is an to.helix, it would be ~350 A long. If, as well, the subunits associate in
60
C. C H O T H I A AND W. L E U Z I N G E R
a parallel manner, ~hrough the globular heads and tails to form the 9 S native r e , t a m e r a n d just through the tails to form the 14 S a n d 18 S native forms, we would h a v e molecules v e r y similar in appearance to those seen in electron micrographs of the native forms. I thank D. M. Blow for making the work possible, and Richard Henderson, Bill Butler and Mary 0shorn for practical help and advice. REFERENCES Berman, J. D. (1973). Biochemistry, 12, 1710-1715. Ben, S., Rieger, F. & Massouli6, J. (1973). Eur. J. Biochem. 35, 372-379. Changeux, J. P., Ryter, A., LeuzJnger, W., Barrand, P. & Podleski, T. (1969). t~roc. Nat. Acad. Sci., U.S.A. 62, 986-993. Chen, Y. T., Rosonberry, T. L. & Chang, H. W. (1974). Arch. Bioehem. Biophys. 151, 479-481. Davis, B. J. (1964). Ann. N . Y . Acad. Sci., 121,404-427. Dudai, Y. & Silman, I. (1971). F E B S Letters, 324-328. Dudai, Y., Silman, I., Kalderon, N. & Blumberg, S. (1972a). Biochim. Biophys. Acta, 268, 138-157. Dudal, Y., Si!m~.u, I., Shinitzky, M. & Blumberg, S. (19725). Prec. Nat. Acad. Sci., U.S.A. 69, 2400-2403. I)udai, Y., Herzberg, M. & Silman, I. (1973). Prec. Nat. Acad. Sci., U.S.A. 70, 2473-2476. Finch, J. T., Crowther, R. A., Hendry, D. A. & Struthers, J. K. (1974). J. Gen. Virol. 24, 191-200. Froede, H. C. & Wilson, I. B. (1970). Israd J. Med. Sci. 6, 179-184. Grafius, M. A. & Millar, D. B. (1965). Bioehim. Biophys. Acta, 110, 540-547. Karnovsky, M. J. & Roots, L. (1964). J. Histoc~em. Cytochem. 12, 219-221. Kremzner, L. T. & Wilson, I. B. (1963). J. Biol. Chem. 238, 1714-1717. Lawler, H. C. (1961). J. Biol. Chem. 236, 2296-2301. Leuzinger, W. (1971). Biochem.J. 123, 139-141. Leuzinger, W. & Baker, A. L. (1967). Prec. 1Vat. Acad. Sci., U.S.A. 57, 446-451. Leuzinger, W., Baker, A. L. & Cauvin, E. (1968). Prec. 2Vat. Acad.Sci., U.S.A. 59, 620-623. Leuzinger, W., Goldberg, M. & Cauvin, E. (1969). J. Mol. Biol. 40, 217-225. Massotfli6, J. & Rieger, F. (1969). Eur. J. Biochem. 11, 441-455. Massouli6, J., Rieger, F. & Tsuji, S. (1970). Eur. J. Biochem. 14, 430-439. Massouli6, J., Rieger, F. & Ben, S. (1971). Eur.J. Biochem. 21, 542-551. Matthews, B. W. (1973). In The ~yroteins, 3 edit. vol. 3, Academic Press, London. Minar, D. B. & Orafius, M. A. (1970). F E B S f_~A~rs, 61-64. Mi||ax, D. B., Grafius, tM. A., Palmer, D. A. & Millar, G. (1973). Eur. J. Biochem. aT, 425-433. 1Kooser, O., Schulman, H. & Sigmars, I). S. (1972). Biochemistry, I1, 1595-1602. Pavlic, M. & Wilson, I.B. (1970). Iugoslav. Physiol. iVha/rmacoL Aeta. 6, 77-81. Powell, J. T., Ben, S., Rieger, F. & Massouli6, J. (1973). I"EBS Letters, 36, 17-22. Rieger, F., Ben, S. & Massouli6, J. (1972). C. R. H. Acad. Sci., Paris, Sdris D, 274, 1753-1756. Rieger, F., Ben, S., Masseur6, J. & Cartaud, J. (1973). Eur. J. Biochem. 34, 539-547. Rosenberry, T. L. & Bernhard, S. A. (1971). Biochemistry, 1O, 4114-4120. Rosenberry, T. L., Chen, Y. T. & Beck, E. (1974). Biochemistry, 13, 3068-3079. Weber, K. & Osborn, M. (1969). J . Biol. Chem. 244, 4406-4412.
Acetylcholinesterase: The Structure o f Crystals o f a Globular F o r m from Electric Eel Cr~us C~oTm,t Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England AND
WALO LEUZ~GER
Instituto de Biofisica, Universidade ]Federal Do Rio de Janeiro, Brazil (Received 30 January 1975, and in revised form 2 May 1975) Acetylcholinesterase prepared from electric eel by the method of Leuzinger & Baker (1967) has a sedimentation coefficient of 11 S and has been described as a "globular" form of the enzyme. Here we report the crystallization of this form and its examination by sodium dodecyl sulphate acrylamide gel eleetrophoresis. The crystals belong to space group P6222 (or P6422) and have the unit cell dimensions a = b ~- 187 A, c = 292 A, y = 120°. The crystal structure and acrylamide gel pattern indicate that the molecule is a tetramer with maximum essential dimensions 93 A × 81 A × 97 ~_. The tail stmmture fmmd in the native (9 S, 14 S and 18 S) forms of the enzyme may be formed by a long s-helix from each subunit. 1. I n t r o d u c t i o n
Nerve impulses are transmitted at cholinergic synapses b y the release of acetylcholine from one cell and its diffusion to a second cell where its interaction with receptors causes depolarization of the potential across the membrane. Also associated with the post-synaptic membrane is acetylcholinesterase which hydrolyses acetylcholine to choline and acetic acid and so terminates the depolarizing action. Acetylcholinesterase (EC 3.1.1.7) has been purified from the electric tissue of Electrophorus electricus b y a variety of methods, most of which initially involve homogenizing the tissue and then storing it in toluene for long periods (Lawler, 1961 ; Kremzner & Wilson, 1963; Leuzinger & Baker, 1967; Massouli~ & Rieger, 1969; Dudai et ed., 1972a) or treating it with trypsin (Dudai et al., 1972a). These methods give five forms of the enzyme in varying proportion (Grafius & Millar, 1965; Massouli6 & Rieger, 1969; Massouli6 eta/., 1970,1971; Dudai e$ a/., 1972a). The five forms can be denoted b y their approximate sedimentation coefficients: three are "native", 18 S, 14 S and 9 S, and two are "globular", 11 S and 8 S. The native forms are distinguished b y their ability to aggregate at low ionic strength (Grafius & MiUar, 1965 ;-Massouli6 & Rieger, 1969; Dudai et al., 1972a) and b y their asymmetric structure (Massouli6 et al., 1971). Present address, to which inquiries concerning this paper should be sent: Service de Bioehimie Cellulaire, Institut Pasteur, 28 rue de Docteur Roux, 75015 Paris, France. 55
56
C. C H O T H I A
A N D W. L E U Z I N G E R
Dudai and Ms colleagues (1972b) purified acetylcholinesterase from electric eel by homogenizing the electric organ and directly passing the supernatant over an af~nlty column, With this method of preparation, only the native 18 S and 14 S forms are obtained in significant quantities. Native forms are converted to globular forms by the action of trypsin (Dudai & Silman, 1971), sonication (Massouli4 et al., 1971) or by allowing solutions to age. This last method involves proteolysis by endogeneous proteases (Rieger et al., 1972). There is good agreement among the values reported for the molecular weight of the 11 S form: 240,000 (Lawler, 1961), 230,000 (Kremzner & Wilson, 1963), 260,000 (Leuzinger et al., 1969), 224,000 (Froede & Wilson, 1970), 240,000 (Pavlic & Wilson, 1970), 260,000 (Miliar & Grafins, 1970); though a value of 320,000 has also been reported (I)udai et al., 1973). The molecular weight of the 18 S form is reported to be 1,100,000 (Bonet a/., 1973; Dudai e~ al., 1973); and the 14 S form to be 780,000 (Bonet al., 1973) and 750,000±150,000 (Dudai et al., 1973). Electron micrographs of the 11 S forms have been published by Changeux et al. (1969), of all five forms by Rieger et a/. (1973) and of the 18 S, 14 S and 11 S forms by I)udai et al. (1973). These workers come to similar conclusions. The 11 S form is seen as a globular particle --~110/~ in diameter with a tetramerie structure formed by subunits each about 50 A in diameter. Occasionally trimers and dimers are seen and are thought to arise from the degradation of tetramers. The native forms however consist of a "head" composed of the 50 A globular subunits and a long semi-rigid "tail". The 9 S molecule has three to four subunits in the head, the tail is ~-~20 A wide and the molecule ~-~500 A long. The head of the 14 S molecule has six to eight subunits and the head of the 18 S molecule at least ten subunits. Both the 14 S and 18 S particles axe about 500 ~ long, and the tails 20 to 30 ~ wide. Rieger et al. (1973) occasionally find short tails, ~100/~, attached to the globular forms. Acetylcholinesterase purified by the method of Leuzinger & Baker (1967) was used in the experiments described here. This form of the enzyme is "globular" (Massouli~ et al., 1971, and see below), has a molecular weight of 260,000 and four subunits (Leuzinger et al., 1969); and a sedimentation coefficient of 11.1 S (Massouli~ et al., 1970). We report here the crystal and subunit structure and molecular dimensions of this form and we discuss the relation of the results to those obtained by other workers who used different techniques.
2. Crystallization and Preliminary X-Ray Data The acetycholinesterase used here had a specific activity of 750 mmol acetylcholine hydrolysed per hour per mg protein. Crystals were grown by the dialysis of a protein solution against solutions of ammonium sulphate. The protein (10 rag/rot in 0-05 •-phosphate buffer, pH 7-4) was placed in small plastic containers which held 5 to 25 pl of solution and had one end open. The open end of the containers was closed with "Visking" dialysis tubing (Scientific Instrument Center Ltd, London) and they were placed in various buffered salt solutions. Salt solutions with 37 to 42~o saturated ammonium sulphate and 0.05 M-phosphate buffer (pH 7.4) gave the best crystals that we obtained. Crystals grew as hexagonal prisms with trigonal termination and are similar in appearance to those obtained by Leuzinger et ed. (1968). Small crystals, 10 to 60 pm long, grew in a few weeks at room temperature and at 4°C. After this, all crystallization vessels were transferred to a cold room (4°C).
ON CRYSTALS OF ACETYLCHOLINESTERASE
57
Several crystals ~100/~m long, grew after a few months and 3, 200 to 300/~n long, after 3 years. X-ray diffraction photographs of the crystals were taken at 4°C on a Nonius Precession camera with an Elliot rotating-anode X-ray tube. At this temperature, crystals in the X-ray beam diffract for about 2 days; at room temperature the diffraction pattern dies very quickly. On "still" photographs, reflections can be seen to a resolution of at least 3.9 A. Plate I shows 2 ° precession photographs of (a) the hk0 plane of the reciprocal lattice and (b) the 0/d plane. The unit cell dimensions are a -~ b ~- 187 A; c ~ 292 A and y ~ 120°. Taken together, the 2 photographs indicate the Laue symmetry of the crystal to be 6]mmol. In the (Okl) zone, the only 001 reflections present are 003, 006 and 009. These results, together with the necessity for an enantiomorphie space group, imply that the space group of the crystals is P6222 (or P6~22). The molecular weight of this form of acetylcholinesterase is 260,000 (Leuzinger et al., 1969). The space group P6222 has 12 general positions. If we assume one molecule per asymmetric unit, the ratio of the volume of the unit cell to the molecular weight of protein in the cell, V~, is 2.6, a common value for protein crystals (Matthews, 1973). Two molecules in the asymmetric unit give a VM value of 1-3 which is unreasonably low and half a molecule in the asymmetric unit gives a V~ value of 5-2 which is unlikely, especially in view of how well the crystals diffract X-rays (Nlatthews, 1973). Thus, it is very likely that these crystals have one molecule in the asymmetric unit. This implies that 53 ~/o of the crystal volume is solvent. In the 0/d diffraction pattern those lines for which l ~- 3n are of greater intensity than those for which 1 ~ 3n. Similarly, on the "still" photographs the rings due to the h~l, hb2 and h/c4 layers are of particularly low intensity. Thus at low resolution the c axis has a pseudo-repeat of c/3 (97/~). The relevance of this point to the shape of the molecule is discussed below.
3. Examination by Acrylamide Gel Electrophoresis The acetylcholinesterase preparation from which the crystals were grown was examined by disc gel electrophoresis following the procedure of Davis (1964). The gels were made of 7.5~/o aerylamide and stained for protein with Coomassie blue and for acetyleholinesterase activity by the direct colouring thiocholine method of Karnovsky & Roots (1964). Electrophoresis of the protein in Tris]glycine buffer, pH 8.3, showed only one component. This component was stained by the direct colouring thiocholine method when acetylthiocholine was used, but not with butylthiocholine, indicating that the enzyme is an acetylcholine specific cholinesterase (Plate II(a)). The protein band was cut out of the gel and, following the procedure of Weber & Osborn (1969), was re-run on a 5~/o acrylamide gel in the presence of sodium dodecyl sulphate and fl-mercaptoethanol, to give the pattern shown in Plate II(b). The same pattern was obtained using the initial protein preparation and carboxymethylated protein. There are two main components: the major has a molecular weight ~58,000 and the minor a molecular weight ~27,000, and traces of other components. The band for the minor component is fairly broad and may be split which indicates some heterogeneity. The molecular weight and subunit structure of this preparation of acetylcholinesterase were previously examined by Leuzinger et al. (1969) using sedimentation equilibrium. They found the molecule to have a molecular weight of 260,000 and to
58
C. C H O T H I A A N D W. L E U Z I N G E R
be c o m p o s e d o f f o u r s u b u n i t s e a c h o f 64,0004-4000. T h e r e was n o t r a c e o f t h e 27,000 m o l e c u l a r w e i g h t p e p t i d e f o u n d h e r e b u t t h e s e a u t h o r s n o t e d t h a t in a p r e l i m i n a r y sedimentation velocity run approximately 15% of the optical density trailed behind t h e m a j o r b o u n d a r y a n d t h a t this f r a c t i o n was r e m o v e d before t h e m o l e c u l a r w e i g h t s were d e t e r m i n e d .
TABLE 1
Molecular weights of the s~b~nits in globular acetylcholinesterase Molecular weight 11 S form 260,000 224,000
8 S form ~ > ~
> 102,000 134,000
1" 320,000
Reference
......
>
-> 160,000
> ~ ÷
t
Subunits 64,0005 49,000 65,000
Leuzinger et al. (19601 Froede & Wilson (1970) Millar et al. (19731
60,000 75,000 64,000§ 88,000
Berman (19731 Dudai & Silman (1971) Dudai et al. (1972a,1073)
70,000][ 58,000¶
Roscnberry et al. (19741 This work
j" Not determined. ~t Same preparation as used here (see text). § In a more recent publication these authors note that their preparations contain peptides of 25,000. II These authors find peptides with a molecular weight of 2%000 and 23,500 (see text). ¶ Peptides of N 27,000 molecular weight also present (see text).
t
c/3 !
J..~3 a 4
FIG. 1. The asymmetric unit of P6~22 or P6422 can be seen as a box a/2, ~/3a/4, c]3 surrounded by 8 2-fold axes. The box drawn here has its centre at z --~ 0. An alternative and equivalent set of boxes can be drawn with their eentres at z = 1/6.
PLATE L 2 ° sereenless precession photographs of eryst~als of acetyleholinesterase. (a) The hk0 reciprocal lattice a n d (b) the O]cZ reciprocal lattice.
[ f ~ i ~ p. 5s
(b) PLATE I•. Aerylamide gel electrophoresis of globular acotyleholinesterase. (a) In Tris/glyeine buffer (pH 8.3) and (b) in sodimn dodecyl sulphate and ~-mercaptoethanoh
ON CRYSTALS OF ACETYLCHOLINESTERASE
59
The subunit-structure of other preparations of 11 S acetylcholinesterase have been investigated by several groups. Their results are summarized in Table 1 and fall into two classes: either molecules have four subunits of molecular weight ,-~60,000 or two of ~60,000 and two of 75,000 to 88,000. (The number of active sites on the 11 S molecule has been reported as 4 (Kremzner & Wilson, 1963; Froede & Wilson, 1970; Rosenberry & Bernhard, 1971; Mooser et al., 1972; Berman, 1973), 3.8 (Chen e~ al., 1974), ~-~3 (Dudai et al., 1972a) and 2 (Leuzinger, 1971).) The heterogeneity of preparations of globular acetylcholinesterase has been examined in detail by Rosenberry and his colleagues (Chen et al., 1974; Rosenberry e$ al., 1974) who conclude that it arises from differing degrees of the proteolytic action involved in converting the native forms of the molecule to the globular forms, Two types of proteolysis occur in the conversion. The first type results in the loss of the tail structure found in the native forms of the enzyme. The second type cleaves the subunits at one or two sites but release of measurable peptides only occurs when the molecules' disulphide bridges are reduced. This second type of proteolysis occurs to a partial and variable extent and produces peptides of 27,000 and 23,500 molecular weight: that is, peptides whose molecular weight is the same as that of the minor component observed here. Inspection of the published sodium dodecyl sulphate acrylamide gel patterns of the native forms of the enzyme (Dudai et al., 1972b, Powell et al., 1973) indicates that they only contain subunits of molecular weight ~-~85,000.
4. Discussion I t would be interesting to know whether the structure seen in the electron micrographs can be related to the crystallographic results. The asymmetric unit of the space group P6222 can be seen as a box, dimensions a[2, ~¢/3a]4, 0]3, surrounded by eight 2-fold symmetry axes (Fig. 1). With the unit cell a ~ b ~ 187 A, c ~ 292 A, this box has the dimensions 93 A × 81 A × 97 A. In the a,b plane the 2-fold symmetry axes which occur at ~ ~ 0, relate adjacent boxes which form a square layer structure. The 62 (or 64) screw axis stacks three of these layers on top of each other to give the unit cell. In this crystal no molecular symmetry coincides with the crystallographic symmetry. Therefore, the molecule cannot cross a 2.fold axis, so that, if the molecule has the globular shape seen in electron micrographs, it must essentially sit inside the asymmetric unit box. This implies that the crystal will have a layer structure and, at low resolution, a pseudo c spacing of c/3. As noted above, the X-ray diffraction pattern at low resolution does have a pseudo spacing of c]3, cleaTly indicating that the crystal has a layer structure. Thus, the crystallographic evidence suggests that this large molecule has a globular shape with the maximum essential dimensions of 93 -~ × 81 A × 97 A. This is a little smaller than the molecule seen in the electron microscope which is ~-~110 A in diameter. This discrepancy is probably due to some flattening of the molecule in the electron microscope (Finch et al., 1974). As noted above, the conversion of the native forms of the enzyme to the globular forms involves the partial or total removal of a ,~ 25,000 molecular weight peptide from each subunit and the concomitant disappearance of the tail structure. I f this peptide is an to.helix, it would be ~350 A long. If, as well, the subunits associate in
60
C. C H O T H I A AND W. L E U Z I N G E R
a parallel manner, ~hrough the globular heads and tails to form the 9 S native r e , t a m e r a n d just through the tails to form the 14 S a n d 18 S native forms, we would h a v e molecules v e r y similar in appearance to those seen in electron micrographs of the native forms. I thank D. M. Blow for making the work possible, and Richard Henderson, Bill Butler and Mary 0shorn for practical help and advice. REFERENCES Berman, J. D. (1973). Biochemistry, 12, 1710-1715. Ben, S., Rieger, F. & Massouli6, J. (1973). Eur. J. Biochem. 35, 372-379. Changeux, J. P., Ryter, A., LeuzJnger, W., Barrand, P. & Podleski, T. (1969). t~roc. Nat. Acad. Sci., U.S.A. 62, 986-993. Chen, Y. T., Rosonberry, T. L. & Chang, H. W. (1974). Arch. Bioehem. Biophys. 151, 479-481. Davis, B. J. (1964). Ann. N . Y . Acad. Sci., 121,404-427. Dudai, Y. & Silman, I. (1971). F E B S Letters, 324-328. Dudai, Y., Silman, I., Kalderon, N. & Blumberg, S. (1972a). Biochim. Biophys. Acta, 268, 138-157. Dudal, Y., Si!m~.u, I., Shinitzky, M. & Blumberg, S. (19725). Prec. Nat. Acad. Sci., U.S.A. 69, 2400-2403. I)udai, Y., Herzberg, M. & Silman, I. (1973). Prec. Nat. Acad. Sci., U.S.A. 70, 2473-2476. Finch, J. T., Crowther, R. A., Hendry, D. A. & Struthers, J. K. (1974). J. Gen. Virol. 24, 191-200. Froede, H. C. & Wilson, I. B. (1970). Israd J. Med. Sci. 6, 179-184. Grafius, M. A. & Millar, D. B. (1965). Bioehim. Biophys. Acta, 110, 540-547. Karnovsky, M. J. & Roots, L. (1964). J. Histoc~em. Cytochem. 12, 219-221. Kremzner, L. T. & Wilson, I. B. (1963). J. Biol. Chem. 238, 1714-1717. Lawler, H. C. (1961). J. Biol. Chem. 236, 2296-2301. Leuzinger, W. (1971). Biochem.J. 123, 139-141. Leuzinger, W. & Baker, A. L. (1967). Prec. 1Vat. Acad. Sci., U.S.A. 57, 446-451. Leuzinger, W., Baker, A. L. & Cauvin, E. (1968). Prec. 2Vat. Acad.Sci., U.S.A. 59, 620-623. Leuzinger, W., Goldberg, M. & Cauvin, E. (1969). J. Mol. Biol. 40, 217-225. Massotfli6, J. & Rieger, F. (1969). Eur. J. Biochem. 11, 441-455. Massouli6, J., Rieger, F. & Tsuji, S. (1970). Eur. J. Biochem. 14, 430-439. Massouli6, J., Rieger, F. & Ben, S. (1971). Eur.J. Biochem. 21, 542-551. Matthews, B. W. (1973). In The ~yroteins, 3 edit. vol. 3, Academic Press, London. Minar, D. B. & Orafius, M. A. (1970). F E B S f_~A~rs, 61-64. Mi||ax, D. B., Grafius, tM. A., Palmer, D. A. & Millar, G. (1973). Eur. J. Biochem. aT, 425-433. 1Kooser, O., Schulman, H. & Sigmars, I). S. (1972). Biochemistry, I1, 1595-1602. Pavlic, M. & Wilson, I.B. (1970). Iugoslav. Physiol. iVha/rmacoL Aeta. 6, 77-81. Powell, J. T., Ben, S., Rieger, F. & Massouli6, J. (1973). I"EBS Letters, 36, 17-22. Rieger, F., Ben, S. & Massouli6, J. (1972). C. R. H. Acad. Sci., Paris, Sdris D, 274, 1753-1756. Rieger, F., Ben, S., Masseur6, J. & Cartaud, J. (1973). Eur. J. Biochem. 34, 539-547. Rosenberry, T. L. & Bernhard, S. A. (1971). Biochemistry, 1O, 4114-4120. Rosenberry, T. L., Chen, Y. T. & Beck, E. (1974). Biochemistry, 13, 3068-3079. Weber, K. & Osborn, M. (1969). J . Biol. Chem. 244, 4406-4412.