J. Mol. Biol. (1991) 218, 675-678
Crystallization and Preliminary X-ray Investigation Recombinant Human Interleukin 4
of
William J. Cook1y4, Steven E. Ealick2p4, Paul Reichert’, Gerald S. Hammond’ Hung V. Le5, Tattanahalli L. Nagabhushan’, Paul P. Trotta’ and Charles E. Bugg3y4 IDepartments of Pathology, 2Pharmacology and 3Biochemistry and 4Center for Macromolecular Crystallography University of Alabama at Birmingham UAB Station, Birmingham, AL 35294, U.S.A. ‘Schering Research BloomJield, NJ 07003, U.S.A. (Received 24 October 1990; accepted 31 December 1990) Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4,2,2 or P4,2,2; the unit cell axes are a = 92.2( 1) A and c = 46.4( 1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63 ye solvent, assuming there is one molecule in the asymmetric unit.
Interleukin 4 (IL-4t) was first identified as a murine cytokine capable of co-stimulating with anti-IgM antibody the proliferation of murine B cells and hence was originally designated B cell stimulating factor-l (Howard et al., 1982). Subsequently, a variety of biological effects of murine IL-4 has been reported, including stimulation of growth of mast cells (Mosmann et al., 1986), thymocytes (Zlotnik et al., 1987) and T cells et al., 1987; (Mosmann et al., 1986; Hu-Li Fernandez-Botran et aZ., 1986), induction of class II major histocompatibility antigens on B cells (Noelle et aZ., 1984; Roehm et al., 1984), enhancement of immunoglobulin production (Vitetta et al., 1985; Coffman & Carty, 1986; Coffman et al., 1986) and both stimulatory and inhibitory effects on hematopoietic progenitor cells (Rennick et al., 1987). On the basis of homology with the murine IL-4 cDNA, a cDNA sequence encoding human IL-4 was isolated (Yokota et al., 1986). Recombinant human IL-4 (rhuIL-4) expressed in mammalian cells was shown to stimulate the proliferation of anti-IgM-activated B cells (Yokota et aZ., 1986; Defiance et al., 1987) and to enhance the proliferation of human T cells and the helper T cell clones (Yokota et al., 1986; Spits et aZ., 1987). In addition, rhuIL-4 enhances the expression on B cell lineages of CD40 (Valle et al., 1989), the low affinity receptor for IgE (Hivroz et
al., 1989; Ishida et al., 1989; Kawabe et al., 1988; Rousset et al., 1988) and lymphocyte function-associated antigens (Rousset et al., 1989), inhibits the expression of the CD5 antigen on B cells (Defiance et al., 1989), stimulates the production of IgG, IgM and IgE (Defiance et al., 1988; Pene et al., 1988) and inhibits the IL-2-dependent proliferation of B-type chronic lymphocytic leukemia cells (Carlsson et al., 1989; Karray et al., 1988). In addition to these effects on lymphocytes, rhuIL-4 promotw the maturation of cells of the myelomonocytic lineage (Grace et al., 1989) and enhances cell-surface antigen expression of mature monocytes (te Velde et al., 1988) and activated neutrophils (Boey et al., 1989). On the basis of these pleiotropic activities, clinical trials with rhuIL-4 have been initiated. The human IL-4 cDNA encodes for a precursor protein composed of 153 amino acid residues including six cyteine residues and two potential sites for N-glycosylation (Yokota et al., 1986). rhuIL-4 has been purified from both mammalian cells (Le et al., 1988) and Escherichia coli (van Amino-terminal al., 1988). Kimmenade et sequencing of rhuIL-4 purified from COS-7 or L-929 cells indicated that the site of processing of the signal sequence occurs between Gly24 and His25 (Le et al., 1988). It has been further established that all six cysteine residues in mammalian cell-derived rhuIL-4 are involved in disulfide bonds, the pairing of which has been established (Windsor et al., 1990). Consistent with the presence of multiple disulfide
t Bbbreviations used: IL-4, interleukin 4; Ig, immunoglobulin; rhuIL-4, recombinant human TL-4. 675 0022%2836/91/080675-04
$03,00/O
0
1991 Academic
Press Limited
676
w. J. Cook et al.
linkages, the E. co&derived rhuIL-4 was found to exhibit high thermodynamic stability (Windsor et al., 1988). We report here the conditions for crystallization of rhuIL-4 derived from E. coli and the results of a preliminary X-ray crystallographic study. Human IL-4 was expressed periplasmically in E. coli (Lundell et al., 1990), with a derivative of the secretory vector pINIIIompA2 (Ghrayeb et al., 1984) and was purified to greater than 98% purity by methodology previously described (Le et al., 1988).
Crystals were initially obtained by vapor-diffusion equilibration of 2-,ul drops hanging from siliconized coverslips inverted on Linbro plates (Flow Laboratories, McLean, VA). The drops consist of 1 cl1 of a solution containing 40 mg protein/ml plus 1 pl of a 50% solution of ammonium sulfate in 605 M-sodium cacody_late buffer. These drops were equilibrated against 1 ml of 50% ammonium sulfate in O-05 M-sodium cacodylate buffer over the pH range 5.0 to 5.8. After one to four days at 22”C, orthorhombic crystals with dimensions up to 0.3 mm x 0.05 mm x 0.05 mm were obtained. Larger crystals generally grew at, the higher pH range. Under similar condit,ions at 12°C no crystals formed after several weeks, but upon placing the drops at 22”C, crystals appeared within one to two days. These drops typically contained fewer crystals, and the crystals were generally larger than those grown only at 22°C. Returning the drops to 12°C initiated dissolution of the crystals. After return of the drops to 22”C, crystals often grew larger t,han their original size. The reappearance of crystals at 22°C occurred irrespective of whether the original crystals were completely or partially dissolved. It appears that the crystals can be cycled between 12°C and 22°C several times, with some improvement in size after each cycle, although the improvement is less after each cycle. Crystals grown by this cycling method grow up to 0.6 mm x 0.1 mm x 0.1 mm. For X-ray studies, crystals were mounted in glass capillaries and were examined with a Nicolet X-100A area detector at 22 “C using CuKa radiation from a Rigaku RU-300 rotating anode generator operating at 40 kV and 100 mA. The detector-tocrystal distance was 16 cm, and the det,ector 28 value was 15”. Oscillation frames covered 025” and were measured for five minutes. A total of 720 frames of data was collected. Indexing and integration of intensity data were carried out using the XENGEN processing computer programs (Howard et al., 1987). Prior to indexing the data, initial estimations of the unit cell constants were determined from several still images. The crystal was oriented for these images such that the various crystal faces were aligned with respect to the incident X-ray beam. The estimations derived from these measurements were used as input to the indexing program. Of the potential solutions generated by the indexing program, one had all angles approximately
equal to 90” and axial lengths consistent with a tetragonal space-group. This was the only solution that successfully indexed all the reflections. The cell parameters refined to a = 92.2(l) A and c = 46*4(l) A (1 A = 61 nm). The unit cell was tested for the two possible tet’ragonal Laue symmetry groups by comparing the integrated intensities of potentially equivalent reflections. This analysis confirmed the Laue group 4/mmm. The systematic absence of reflections hO0 with h # 2w and 001 with I # 4n indicates either space-group P4,2,2 or its enantiomorph P4,2,2. The use of electronic area detectors to determine space-groups has a number of advantages over conventional film methods. These include the following. (1) X-ray patterns can be measured much more rapidly using an electronic area detector. (2) No pre-alignment of the crystal is required. (3) This method utilizes three-dimensiona,i data two-dimensional zones. rather than special (4) Computerized analysis of integrated intensities is more reliable than visual estimation of spot densities. (5) Intensities of potential systematic a.bsenc~es can be compared to their standard deviations. (6) The analysis provides a means for quantitatively assessing the limit of resolution. (7) The process ifan result in a complete three-dimensional data set. There are several disadvantages: (1) the detector-tocrystal distance must be large enough to resolve reflections for the unknown unit cell. (2) The pot,ential for selecting an incorrect space-group is greatet than with systematic film methods because of reliance on automated indexing routines. For example, weak intensities may be overlooked by the programs that select, reflections for input- to t’he indexing routines. The omission of even a few we& reflections may prevent the identification of the correct symmetry or unit cell parameters. This problem may be overcome by using computer graphics to compare t,he calculated patterns with the observed pat,terns for individual frames of data and by the statistical analysis of groups of symmetry-related intensities for all predicted reflections. (3) The initial area detector data may not include observations for all possible systema,tic absences, resulting in incorrect identification of space-group symmetry. Therefore, final determination of systematic absences should be based on comparison of the integrated intensities with their standard deviations at the predicted reflection positions for a complete data set. As electronic area detectors become available to more users, this method of space-group determination will proba,blg be used more widely. As wit,11 precession film methods, careful examination of the area detecbtor intensities should ensure that the correct spaeegroup is identified. A complete native dat,a set to 2.8 A resolution was collected. A total of 25,627 reflections was processed; these were merged into 5191 unique reflections. The R,,, value (based on I) for the dat,a to 28 A was @072. Of the 876 reflect’ions in the highest resolution range (2.8 to 3.0 A), 713 reflex:-
Communications tions (81%) had I > 301. The crystals are stable to X-rays at room temperature for at least three days and diffract beyond 2.8 A resolution. On the basis of a molecular weight of 14,964 daltons as predicted from the cDNA (Yokota et al., 1986), the calculated value of V, (Matthews, 1968) for one molecule/ crystallographic asymmetric unit is 3.29 A3/dalton. Assuming a partial-specific-volume of 074 ml/g, this corresponds to a solvent volume fraction of 63%. These data have provided a basis for X-ray crystallographic analysis of the structure of rhuIL-4, which is currently in progress. We gratefully acknowledge the contribution of Molecular Biology and Research and Development at Schering-Plough Research for the expression and purification of recombinant human IL-4. We thank Leigh Jeffrey for technical assistance. Research supported by grant NAGW813 from the National Aeronautics and Space Administration, grants CA-13148 and DE-08228 from the Xational Institutes of Health, grant CH-213 from the American Cancer Society, and a grant from the National Foundation for Cancer Research.
References Boey, H., Rosenbaum, R., Castracane, J. & Borish, L. (1989). Interleukin-4 is a neutrophil activator. J. Allergy
Clin.
Immunol.
83, 978-984.
Carlsson, M., Sundstriim, C., Bengtsson, M., Totterman, T. H., Rosen, A. & ru’ilsson, K. (1989). Interleukin 4 strongly augments or inhibits DPU’A synthesis and differentiation of B-type chronic lymphocytic leukemia cells depending on the co-stimulatory activation and progression signals. Eur. J. Immunol. 19; 913-921. Coffman, R. L. & Carty, J. (1986). A T cell activity that enhances polyclonal IgE production and its inhibition by interferon-y. J. Immunol. 136, 949-954. Coffman, R. L., Ohara, J., Bond, M. W., Carty, J., Zlotnik, A. & Paul, W. E. (1986). B cell stimulatory factor-l enhances the IgE response of lipopolysacB cells. J. Immunol. charide-activated 136, 453884541. Defiance, T., Vanbervliet, B., Aubrey, J.-P., Takabe, Y., Arai, Tu’., Miyajima, A., Yokota, T., Lee, F., Arai, K.-I., de Vries, J. E. & Banchereau, J. (1987). B cell growth-promoting activity of recombinant human interleukin 4. J. Immunol. 139, 1135-1141. Defiance, T., Vanbervliet, B., P&e, J. & Banchereau, J. (1988). Human recombinant IL-4 induces activated B lymphocytes to produce IgG and IgM. J. Immunol. 141, 2000-2005. Defiance, T.; Vanbervliet, B., Durand, I. & Banchereau, J. (1989). Human interleukin 4 down-regulates the surface expression of CD5 on normal and leukemic B cells. Eur. J. Immunol. 19, 293-299. Fernandez-Botran, R., Krammer, P. H., Diamantstein, T., Uhr, J. W. & Vitetta, E. S. (1986). B cell-stimulatory factor 1 (BSF-1) promotes growth of helper T cell lines. J. Exptl. Med. 164, 580-593. Ghrayeb, J., Kimura, H., Takahara, M., Hsiung, H., Masui, Y. & Inouye, M. (1984). Secretion cloning vectors in Escherichia coli. EMBO J. 3, 2437-2442. Grace, M. J., Bober, L. H., Bennett, B. F., Simpson, E. H. & Waters, T. A. (1989). Human recombinant IL-4 helps promote differentiation of U-937 promonocytic
617
and
HL-60
cell lines. Proc. 7th p. 213 (Abstr. 38-8). Hivroz, C., Valle, A., Brouet, J. C., Banchereau, J. & Grillot-Courvalin, C. (1989). Regulation by interleukin 2 of CD23 expression of leukemic and normal B cells: comparison with interleukin 4. Eur. J. Immunol. 19, 1025-1030. Howard, A. J., Gilliland, G. L., Finzel, B. C., Poulos, T. L., Ohlendorf, D. H. & Salemme, F. R. (1987). The use of an imaging proportional counter in macromoJ. Appl. Crystallogr. 20, lecular crystallography. 383-387. Howard, M., Farrar, J.; Hilfiker, M., Johnson, B., Takatsu, K.; Hamaoka, T. &. Paul, W. E. (1982). Identification of a T cell-derived B cell growth factor Med. 155, distinct from interleukin 2. J. Exptl. 914-923. Hu-Li, J., Shevach, E. M., Mizuguchi, J., Ohara, J., Mosmann, T. & Paul, W. E. (1987). B cell stimulatory factor 1 (interleukin 4) is a potent costimulant for normal resting T lymphocytes. J. Exptl. Med. 165, 157-172. Ishida, H., Kumagai, S.; Iwai, K., Takahashi, T., Kawabe, T.; Yodoi, J., Arai, pu’., Namiuchi, S., Konaka, Y. & Imura, H. (1989). Heterogeneity in terms of interleukin 4-dependent regulation of FCE receptor/CD23 expression on chronic B-lymphocytic leukemia cells. ImmunoZ. Letters, 20, 323-330. Karray, S.; Defiance, T., Merle-B&al, H., Banchereau, J., Deb&, P. & Galanaud, P. (1988). Interleukin 4 counteracts the interleukin S-induced proliferation of monoclonal B cells. J. Exptl. Med. 168: 85-94. Kawabe, T., Takami, M., Hosoda, M., Maeda, Y., Sato, S., Mayumi; M., Mikawa, H., Arai, K.-I. & Yodoi, J. (1988). Regulation of Fe&R&D23 gene expression by cytokines and specific ligands (IgE and anti-FcsR, monoclonal antibody). J. Immunol. 141, 1376-1382. Le, H. V., Ramanathan, L., Labdon, J. E., Mays-Ichinco, C. A., Syto, R., Arai, N., Hoy, P.; Takebe, Y., Nagabhushan, T. L. & Trotta, P. P. (1988). Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells. J. Biol. Chem. 263, 10817-10823. Lundell, D.; Greenberg, R., Alroy; Y., Condon, R., Fossetta, J. D., Gewain, K., Kastelein, R., Lunn, C. A., Reim, R., Shah, C., van Kimmenade, A. & Narula, S. K. (1990). Cytoplasmic and periplasmic expression of a highly basic protein, human interleukin 4, in Escherichia coli. J. Indust. Micro. 5, 215-228. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33; 491-497. Mosman, T. R., Bond, M. W., Coffman, R. L.; Ohara, J. & Paul, W. E. (1986). T cell and mast cell lines respond to B cell stimulatory factor 1. Proc. Nat. Acad. Sci., Internat.
U.S.A.
premyelocytic
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83, 5654-5658.
Noelle, R., Krammer, P. H., Ohara, J.; Uhr, J. W. & Vitetta, E. S. (1984). Increased expression of Ia antigens on resting B cells: an additional role for B cell growth factor. Proc. Nat. Acad. Sci., U.S.A. 81, 6149-6153. P&e, J., Rousset, I?., Briere, F., Chretien, I., Bonnefoy, J.-Y., Spits, H., Yokota, T.; Arai, N., Arai. K.-I., Banchereau, J. & de Vries; J. E. (1988). IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons y and CI and prostaglandin E,. Proc. Nat. Acad. Sci., U.S.A. 85, 688066884.
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678
Rennick, D., Yang, G., Muller-Sieburg: C., Smith, C., Arai, h’., Takabe, Y. & Gemmell, L. (1987). Interleukin 4 (B-cell stimulatory factor 1) can enhance or antagonize the factor-dependent growth of hemopoietic progenitor cells. Proc. Nat. Acad. Sci., U.S.A. 84, 6889-6893. Roehm, N. W.; Leibson, H. J., Zlotnik, A., Kappler, J., Marrack, P. & Cambier, J. C. (1984). Interleukininduced increase in Ia expression by normal mouse B cells. J. Exptl. Med. 160, 679-694. Rousset, F., de Waal Malefijt, R., Slierendregt, B., Aubry, J.-P., Bonnefoy, J.-Y.; Defiance, T., Banchereau, J. & de Vries, J. E. (1988). Regulation of Fe receptor for IgE (CD23)-and class II MHC a,ntigen expression on Burkitt’s lymphoma cell lines by human IL-4 and IFE-y. J. Immunol. 140, 2625-2632. Rousset, F., Billaud, -VI., Blanchard, D.. Figdor. C., Lenoir, G. M.. Spits, H. & de Vries, J. E. (1989). TL-4 induces LFA-1 and LFA-3 expression on Burkitt’s lymphoma cell lines. J. Immunol. 143, 1490-1498. Spits, H., Yssel,-II., Takebe, Y., Arai. X., Yokota. T.. Lee, F., Ara.i, K.-I., Banchereau, J. & de Vries. *J. E. (1987). Recombinant interleukin 4 promot,es the of human T cells. J. Immunol. 139. growth 1142-1147.
te Velde, il. A., Klomp, J. I?. G., Yard, B. A.. de Vrirs, J. E. & Figdor, C. G. (1988). Modulation of phenotypic and functional properties of human peripheral blood monocytes by IL-4. J. Immunol. 140. 1548-1554. VallB, A., Zuber, C. E., Defiance, T., Djossou, O.,‘de Rie. M. & Banchereau, J. (1989). Activation of human 15 lymphocytes through CD40 and interleukin 4. Eur. J. Immu,nol. 19, 1463-1467.
van Kimmenade; A., Bond, M. W., Schumacher. ,1. Fi.. Laquoi, C. & Kastelein, R. A. (1988). Expression. renaturation and purification of recombinant huma,n interleukin 4 from Escherichia coli. Ew. .J. Wio~hrm. 173, 109-I 14. Vitetta, E. S., Ohara, J.. Myers, C. I)., Layton. .J. ti:., Krammer. P. H. & Paul, W. E. (1985). Serological. biochemical. and funct,ional identity of B c*e:li-stirnillatory fact,or 1 and B cell differentiation factor for HgGl. J. Exptl. Med. 162, 1726-1531. Windsor, W. T., Syto, R.. Nagabhushan. T. T,.. Trot’ta. P. P. & Lee, H. V. (1988). Conformation and stahilit) 4 (huTL1). of recombinant human interleukin FASEB J. 2, Al337 (Abstr. 6052). Windsor, W. T., Syto, R’., Durkin, *I.. Das, I’., Rrichprt. I’.; Pramanik, B., Tindall, S.. Le, N. V.. I,abdon. J.. Nagabhushan, T. L. & Trotta, P. P. (1990). IXsulfidr bond assignment of mammalian cell-derived recombnant human interleukin 4. Biophys. J. 57, 423a (Abstr. W-Pos41). Yokota: T., Otsuka, T., Mosmann, T.. Banchereau, J.. Defiance, T.; Blanchard, I).. de Vries. .J. E., Lee, F. & Arai; K.-I. (1986). Isolation and characterization of a human interleukin cDIV4 clone, homologous to mouse B cell stimulatory faact,or I. t,hat expresses B-cell- and T-cell-stimulating activities. f'roc. Sat. ilcad.
Sci., C:.S.il.
83. 58944898.
Zlot’nik, A., Ransom, J,, Frank, G.. Fischer. JI. 8r Howard, M. (1987). Interleukin 4 is a, growt,h iac.tol. for activated thymocytes: possible role in T-cell ontogeny. Proc. Kat. Acad. Scci., U.S.A. 84. 38;56-3860.
Edited by B. $1. #atthews
Crystallization and Preliminary X-ray Investigation Recombinant Human Interleukin 4
of
William J. Cook1y4, Steven E. Ealick2p4, Paul Reichert’, Gerald S. Hammond’ Hung V. Le5, Tattanahalli L. Nagabhushan’, Paul P. Trotta’ and Charles E. Bugg3y4 IDepartments of Pathology, 2Pharmacology and 3Biochemistry and 4Center for Macromolecular Crystallography University of Alabama at Birmingham UAB Station, Birmingham, AL 35294, U.S.A. ‘Schering Research BloomJield, NJ 07003, U.S.A. (Received 24 October 1990; accepted 31 December 1990) Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4,2,2 or P4,2,2; the unit cell axes are a = 92.2( 1) A and c = 46.4( 1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63 ye solvent, assuming there is one molecule in the asymmetric unit.
Interleukin 4 (IL-4t) was first identified as a murine cytokine capable of co-stimulating with anti-IgM antibody the proliferation of murine B cells and hence was originally designated B cell stimulating factor-l (Howard et al., 1982). Subsequently, a variety of biological effects of murine IL-4 has been reported, including stimulation of growth of mast cells (Mosmann et al., 1986), thymocytes (Zlotnik et al., 1987) and T cells et al., 1987; (Mosmann et al., 1986; Hu-Li Fernandez-Botran et aZ., 1986), induction of class II major histocompatibility antigens on B cells (Noelle et aZ., 1984; Roehm et al., 1984), enhancement of immunoglobulin production (Vitetta et al., 1985; Coffman & Carty, 1986; Coffman et al., 1986) and both stimulatory and inhibitory effects on hematopoietic progenitor cells (Rennick et al., 1987). On the basis of homology with the murine IL-4 cDNA, a cDNA sequence encoding human IL-4 was isolated (Yokota et al., 1986). Recombinant human IL-4 (rhuIL-4) expressed in mammalian cells was shown to stimulate the proliferation of anti-IgM-activated B cells (Yokota et aZ., 1986; Defiance et al., 1987) and to enhance the proliferation of human T cells and the helper T cell clones (Yokota et al., 1986; Spits et aZ., 1987). In addition, rhuIL-4 enhances the expression on B cell lineages of CD40 (Valle et al., 1989), the low affinity receptor for IgE (Hivroz et
al., 1989; Ishida et al., 1989; Kawabe et al., 1988; Rousset et al., 1988) and lymphocyte function-associated antigens (Rousset et al., 1989), inhibits the expression of the CD5 antigen on B cells (Defiance et al., 1989), stimulates the production of IgG, IgM and IgE (Defiance et al., 1988; Pene et al., 1988) and inhibits the IL-2-dependent proliferation of B-type chronic lymphocytic leukemia cells (Carlsson et al., 1989; Karray et al., 1988). In addition to these effects on lymphocytes, rhuIL-4 promotw the maturation of cells of the myelomonocytic lineage (Grace et al., 1989) and enhances cell-surface antigen expression of mature monocytes (te Velde et al., 1988) and activated neutrophils (Boey et al., 1989). On the basis of these pleiotropic activities, clinical trials with rhuIL-4 have been initiated. The human IL-4 cDNA encodes for a precursor protein composed of 153 amino acid residues including six cyteine residues and two potential sites for N-glycosylation (Yokota et al., 1986). rhuIL-4 has been purified from both mammalian cells (Le et al., 1988) and Escherichia coli (van Amino-terminal al., 1988). Kimmenade et sequencing of rhuIL-4 purified from COS-7 or L-929 cells indicated that the site of processing of the signal sequence occurs between Gly24 and His25 (Le et al., 1988). It has been further established that all six cysteine residues in mammalian cell-derived rhuIL-4 are involved in disulfide bonds, the pairing of which has been established (Windsor et al., 1990). Consistent with the presence of multiple disulfide
t Bbbreviations used: IL-4, interleukin 4; Ig, immunoglobulin; rhuIL-4, recombinant human TL-4. 675 0022%2836/91/080675-04
$03,00/O
0
1991 Academic
Press Limited
676
w. J. Cook et al.
linkages, the E. co&derived rhuIL-4 was found to exhibit high thermodynamic stability (Windsor et al., 1988). We report here the conditions for crystallization of rhuIL-4 derived from E. coli and the results of a preliminary X-ray crystallographic study. Human IL-4 was expressed periplasmically in E. coli (Lundell et al., 1990), with a derivative of the secretory vector pINIIIompA2 (Ghrayeb et al., 1984) and was purified to greater than 98% purity by methodology previously described (Le et al., 1988).
Crystals were initially obtained by vapor-diffusion equilibration of 2-,ul drops hanging from siliconized coverslips inverted on Linbro plates (Flow Laboratories, McLean, VA). The drops consist of 1 cl1 of a solution containing 40 mg protein/ml plus 1 pl of a 50% solution of ammonium sulfate in 605 M-sodium cacody_late buffer. These drops were equilibrated against 1 ml of 50% ammonium sulfate in O-05 M-sodium cacodylate buffer over the pH range 5.0 to 5.8. After one to four days at 22”C, orthorhombic crystals with dimensions up to 0.3 mm x 0.05 mm x 0.05 mm were obtained. Larger crystals generally grew at, the higher pH range. Under similar condit,ions at 12°C no crystals formed after several weeks, but upon placing the drops at 22”C, crystals appeared within one to two days. These drops typically contained fewer crystals, and the crystals were generally larger than those grown only at 22°C. Returning the drops to 12°C initiated dissolution of the crystals. After return of the drops to 22”C, crystals often grew larger t,han their original size. The reappearance of crystals at 22°C occurred irrespective of whether the original crystals were completely or partially dissolved. It appears that the crystals can be cycled between 12°C and 22°C several times, with some improvement in size after each cycle, although the improvement is less after each cycle. Crystals grown by this cycling method grow up to 0.6 mm x 0.1 mm x 0.1 mm. For X-ray studies, crystals were mounted in glass capillaries and were examined with a Nicolet X-100A area detector at 22 “C using CuKa radiation from a Rigaku RU-300 rotating anode generator operating at 40 kV and 100 mA. The detector-tocrystal distance was 16 cm, and the det,ector 28 value was 15”. Oscillation frames covered 025” and were measured for five minutes. A total of 720 frames of data was collected. Indexing and integration of intensity data were carried out using the XENGEN processing computer programs (Howard et al., 1987). Prior to indexing the data, initial estimations of the unit cell constants were determined from several still images. The crystal was oriented for these images such that the various crystal faces were aligned with respect to the incident X-ray beam. The estimations derived from these measurements were used as input to the indexing program. Of the potential solutions generated by the indexing program, one had all angles approximately
equal to 90” and axial lengths consistent with a tetragonal space-group. This was the only solution that successfully indexed all the reflections. The cell parameters refined to a = 92.2(l) A and c = 46*4(l) A (1 A = 61 nm). The unit cell was tested for the two possible tet’ragonal Laue symmetry groups by comparing the integrated intensities of potentially equivalent reflections. This analysis confirmed the Laue group 4/mmm. The systematic absence of reflections hO0 with h # 2w and 001 with I # 4n indicates either space-group P4,2,2 or its enantiomorph P4,2,2. The use of electronic area detectors to determine space-groups has a number of advantages over conventional film methods. These include the following. (1) X-ray patterns can be measured much more rapidly using an electronic area detector. (2) No pre-alignment of the crystal is required. (3) This method utilizes three-dimensiona,i data two-dimensional zones. rather than special (4) Computerized analysis of integrated intensities is more reliable than visual estimation of spot densities. (5) Intensities of potential systematic a.bsenc~es can be compared to their standard deviations. (6) The analysis provides a means for quantitatively assessing the limit of resolution. (7) The process ifan result in a complete three-dimensional data set. There are several disadvantages: (1) the detector-tocrystal distance must be large enough to resolve reflections for the unknown unit cell. (2) The pot,ential for selecting an incorrect space-group is greatet than with systematic film methods because of reliance on automated indexing routines. For example, weak intensities may be overlooked by the programs that select, reflections for input- to t’he indexing routines. The omission of even a few we& reflections may prevent the identification of the correct symmetry or unit cell parameters. This problem may be overcome by using computer graphics to compare t,he calculated patterns with the observed pat,terns for individual frames of data and by the statistical analysis of groups of symmetry-related intensities for all predicted reflections. (3) The initial area detector data may not include observations for all possible systema,tic absences, resulting in incorrect identification of space-group symmetry. Therefore, final determination of systematic absences should be based on comparison of the integrated intensities with their standard deviations at the predicted reflection positions for a complete data set. As electronic area detectors become available to more users, this method of space-group determination will proba,blg be used more widely. As wit,11 precession film methods, careful examination of the area detecbtor intensities should ensure that the correct spaeegroup is identified. A complete native dat,a set to 2.8 A resolution was collected. A total of 25,627 reflections was processed; these were merged into 5191 unique reflections. The R,,, value (based on I) for the dat,a to 28 A was @072. Of the 876 reflect’ions in the highest resolution range (2.8 to 3.0 A), 713 reflex:-
Communications tions (81%) had I > 301. The crystals are stable to X-rays at room temperature for at least three days and diffract beyond 2.8 A resolution. On the basis of a molecular weight of 14,964 daltons as predicted from the cDNA (Yokota et al., 1986), the calculated value of V, (Matthews, 1968) for one molecule/ crystallographic asymmetric unit is 3.29 A3/dalton. Assuming a partial-specific-volume of 074 ml/g, this corresponds to a solvent volume fraction of 63%. These data have provided a basis for X-ray crystallographic analysis of the structure of rhuIL-4, which is currently in progress. We gratefully acknowledge the contribution of Molecular Biology and Research and Development at Schering-Plough Research for the expression and purification of recombinant human IL-4. We thank Leigh Jeffrey for technical assistance. Research supported by grant NAGW813 from the National Aeronautics and Space Administration, grants CA-13148 and DE-08228 from the Xational Institutes of Health, grant CH-213 from the American Cancer Society, and a grant from the National Foundation for Cancer Research.
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