J. Mol. Biol. (1975) 97, 395-397

Characterization of Crystals of Two Venom Phospholipases A~ Crystals of phosphohpase A= from Grotal~ adama~eu.~ and C. at~oz have been grown which aresuitable for X-ray diffraction analysis to at least 2.8 ~ and 2.2 ~, respectively. Phospholipase A=¢t from G. a ~ a r n a n ~ crystallizes in ,space group C222z, a = 108-1 ~, b = 79.4 ~, o = 63.9 ~, 30,000 molecular weight (one dimer) per asymmetric unit. Phospholipase A= from C. at~oz crystallizes in space group P2z2x2z, a = 53.8 ~, b = 99-0 .~, o = 49.5 ~, 29,760 molecular weight (one dimer) per asymmetric unit. The phospholipases A2 are enzymes which catalyze the cleavage of 2-fatty acyl groups from 3-gn-phosphoglycerides. They have been isolated from a variety of sources, including pancreatic tissue of man (BeUeville & Clement, 1968), pig (De Haas et at., 1968) and rat (Arnesj5 et aL, 1967); bee venom (Shipolini et ¢1., 1971); and several snake venoms (Wells & Hanahan, 1969; Hachimori et a/., 1971). These proteins are central to studies in phospholipid structure and metabolism, and, in addition they m a y provide a useful model for other types of protein-phospholipid interactions. The protomer of phospholipase A 2 from Crotal~s a d ~ z n t e ~ s (Eastern diamondback rattlesnake) is a chain of 135 amino acids and has a molecular weight of 15,000, while the protomer of phospholipase A 2 from Crotal~ atrox (Western diamondback rattlesnake) has a molecular weight of 14,880 and consists of 134 amino acids (unpublished data B. W. Shen). Each protomer possesses 14 half-cystine residues, a rather high percentage. Both proteins bind calcium ions as cofactor and both are functional only as dimers; t h a t is, the protomer in each case is catalytically inactive (Wells, 1971; Shen ~ ¢1., 1975; Hachimori ~ al., 1971). This phenomenon contrasts with that of a large class of phospholipases A~, including those from other snake venoms and t h a t from porcine pancreas, in which the monomer is the active species (Wells, 1971). This difference in behavior is rather surprising, in view of the sequence homologies observed between these various phospholipases (Tsao et al., 1975). A model of the protomer-protomer interface is needed to identify those structural features essential to this productive interaction between subunits. Worlr is in progress on the crystal structures of mammalian pancreatic phospholipases in the lab of Professor J a n Drenth at GrSningen University. Structural information about the active site regions of several of the venom phospholipases A2, including t h a t from C. a d a m a ~ e ~ , is available from amino acid sequence data (Tsao st al., 1975, and references therein). We report here a pre]imlnary X-ray crystallographic study of large single crystals of phospholipase A~ from C. adama~eu8 and from G. c~rox. Photographs of crystals of both phospholipases A2 are shown in Plate I ((a) and (b)). Both proteins crystallize spontaneously over a period of 24 to 72 hours under identical t Phospholipase A= from (7. adamar~st~sexist~ in two chromatographically distinct forms, = and /] (Wells & Hanahau, 1969). The chemical differences are not auderstood. The results below are with phospholipase A==. Although wi~h phospholipase A=~ the described cryst~|]i~.stionprotocol yields orysf~is in the shape of thin plates exoesrllngone m~ll~meteron an edge, no characterization of these crystals is available at this time. 395

396

lV[. P A S E K , C. K E I T H , D. FELDMAN AND P. B. S I G L E R

conditions. A solution of 9 mg protein/ml, 10 m~-sodium cacodylate (pH 6.4), 1 mM-CaC12, and 10 raM-polyethylene glycol (Baker; 6000 to 7500 molecular weight) was pipetted-in small aliquots of 20 microliters in the concavities of depression slides coated with Siliclad (Clay-Adams), the slides were placed in plastic boxes over a 25 ml reservoir of 10 raM-polyethylene glycol, and the boxes sealed. I n general, crystals of a millimeter or more along the b axis were readily obtained. The quality of crystals of phospholipase A2 from C. atrox is particularly sensitive to the calcium ion concentration; large crystalline aggregates of more than one visually distinct domain grow under conditions identical to those above, but with a calcium ion concentration of 10 raM. The diffraction patterns obtained from these crystals are shown in Plate I ((c) to (f)). I n the case of phospholipase A2 from C. adamanteus, strong diffraction maxima are clearly evident to 2.8 A resolution (Plate I(c) and (e)). Diffraction photographs of crystals of phospholipase A2 from C. atrox show strong reflections to 2-8 A resolution (Plate I(d) and (f)) and preliminary oscillation photographs taken on an oscillation camera built in this laboratory b y Gary Cornick show that the pattern extends to at least 1.8 A resolution. The diffraction pattern of these crystals of phospholipase A2 from C. atrox starts to show a visually apparent decrease in the intensities of reflections at 2.8 A resolution only after 60 hours X-irradiation (Ni-filtered CuKa, 40 kV, 30 mA). Table 1 compares the properties of crystals of phospholipases A2 from C. adamanteus and C. atrox, including principal unit cell translations, unit cell volumes, and dimers per asymmetric unit. Space groups were assigned on the basis of systematic absences in the axial projections shown in Plate I. I t is worthwhile to note that the unit cell volumes of the two crystals differ b y a factor of two to within 4 % and t h a t the unit cell dimension along the a axis differs b y a factor of 2. We are unable, however, to derive from a consideration of these data a description in structural terms of the contacts between monomers which are important in lattice construction for each crystal. I t is also ditt~cult to see any relation between the intensity distributions of TABLE 1

Comparison of ¢rystal~ of phospholipases A~ from C. adamanteus and C. atrox (J. adarnanteus Space group a (A) b (A) c (/k)

Density of crystal (g/cmS)t Volume fraction of protein~ Daltons of protein per asymmetric unit~ Unit cell volume (A3) IZm (Aa per dalton)~;

C2221 108.1 79-4

63.9 1.208 0.540 30,000 548,000 2-28

C. atrox P212121 53.8 99.0

49.5 1.228 0.598 29,760 264,000 2"21

t Measured as described in Corniek et aL (1971). Determined assuming a value of 0.722 em3/g for the partial racial volume (17) for phospholipase A~ from C. adarnanteu~and 0.724 emS/g for that for phospholipase Az from C. atrox (unpublished data courtesy of B. W. Shen). The density of protein-free liquid of crystal|iT.ation, 10 mMsodium cacodylate (pH 6.40)-1 n~'-CaC12-10 raM-polyethylene glycol, was assumed to be 1.000 g/em a.

PLATE I. Crystals of phospholipases A2 from C. adamanteua a n d C. atrox a n d their diffraction patterns. (a) Crystal Of phospholipase A2~ from C. adamanteua. (b) Crystal of phospholipase A2 from C. atrox. (c) a n d (e) are two principal zones, (Obl) a n d (hk0), respectively, obtained from crystals of phospholipaso A2c~from C. adamanteua; space group 0222z. (d) a n d (f) are two principal zones, (Okl) a n d (hk0), respectively, obtainecl from crystals of phospholipase A2 from C. atrox; space group P2z2z2z. P h o t o g r a p h s (e), (d), (e), a n d (f) were obtained w i t h a crystal-to-film plane distance 75 m m a n d precession angle 16 ° after 48, 48, 48 a n d 24 hours exposure, respectively, to Ni-filtered CuKot radiation from a I~orelco fine-focus X - r a y tube operated a t 40 kV a n d 30 mA. [facinq ~. 396

LETTERS

TO THE

EDITOR

397

these two crystals. I t is quite plausible t h a t the dimer of the asymmetric unit is the functional dimer, particularly in the crystals of O. atrox where there are no lattice dyads and where it is unlikely protomers in a functional dimer are related b y crystallographic screw axes. A rotation and translation search (Rossmann & Blow, 1962; Rossmann et al., 1964) would establish the relation between the protomers within the asymmetric unit. This information could be used (a) to facilitate the interpretation of h e a v y - a t o m derivatives; (b) to exploit the redundancy of structure within the asymmetric unit as an aid to improving the accuracy of the electron density m a p ; and (c) possibly to identify a physiologically i m p o r t a n t interface between protomers. The authors are grateful to Drs R. L. Heinrikson, F. J. K~zdy, and J. H. Law, Department of Biochemistry, for valuable discussions during the course of this work, and for generous gifts of phospholipases Am from G. adamanteu~ and G. atrox. This work has been supported by National Institutes of Health research grant GM15525 and by National Science Foundation research grant BMS74-15075. Two of us (C. K. and M. P.) are United States Public Health Service predoctoral trainees (grant nos. GM780 and GM424, respectively). Departments of Biochemistry and Biophysics and Theoretical Biology Cummings Life Science Center The University of Chicago Chicago, Ill. 60637, U.S.A.

MARK PASEK

CHARLES KEITH t DAVID FELDMA ~T

PAUL B. SINLESS

Received 21 April 1975 REFERENCES ArnesjS, B., Barrowman, J. & BorgstrSm, B. (1967). Actu Ohem. Scand. 21, 2897-2900. BeUeville, J. & Cldment, J. (1968). Bull. Soc. Ohim. Biol. 50, 1419-1424. Cornick, G., Sigler, P. B. & Ginsberg, H. S. (1971). J. Mol. Biol. 57, 397-401. De Haas, G. H., Postema, N. M., Nieuwenhuizen, W. & Van Deenen, L. L. M. (1968). Biochlm. Biophys. Acta, 159, 103-117. Hachimori, Y., Wells, M. A. & Hanahan, D. J. (1971). Biochemistry, 19, 4084-4088. Rossmann, M. G. & Blow, D. M. (1962). Acta Grys~llogr. 15, 24-31. Rossmann, M. G., Blow, D. M., Harding, M. M. & Coller, E. (1964). Acta Grystallogr. 17, 338-342. Shen, B. W., Tsao, F. H. C., Law, J. H. & Kdzdy, F. J. (1975). J. Amer. Ghem. Soc. 97, 1205-1208. Shipolini, R. A., Callewaert, G. L., Cottrell, R. C., Doonan, S., Vernon, C. A. & Banks, B. E. C. (1971). Eur. J. Biechem. 2{), 459-468. Tsao, F. H. C., Keim, P. S. & Heinrikson, R. L. (1975). Arch. Biochem. Biophys. 167, 706-717. Wells, M. A. (1971). Biochemistry, 1@, 4074-4078. Wells. M. A. & Hanahan, D. J. (1969). Biochemistry, 8, 414-424.

$ To whom correspondenee should be addressed. On sabbatical leave. Present address: Department of Structural Chemistry, Weizmann Institute, Rehovot., Israel.

Characterization of crystals of two venom phospholipases A2.

J. Mol. Biol. (1975) 97, 395-397 Characterization of Crystals of Two Venom Phospholipases A~ Crystals of phosphohpase A= from Grotal~ adama~eu.~ and...
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