PROTEINS: Structure, Function, and Genetics 12:24-30 (1992)
Crystallization Studies of Glycosylated and Unglycosylated Human Recombinant Interleukin-2 Enrico A. Stura, Ping Chen, Carrie M. Wilmot, Jairo H. Arevalo, and Ian A. Wilson Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
ABSTRACT Glycosylated interleukin-2 (glyIL-2) has been crystallized in two crystal forms, and unglycosylated interleukin-2 (uIL-2) has been crystallized in three forms. The glycosylated form of the human recombinant IL-2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space group P6,22 or its enantiomorph. The crystals diffract to 2.8 A and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0-5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL-2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.55.6 in the monoclinic space group P2,, and less frequently in the orthorhombic space group P2,2,2, from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross-seeding uIL-2 with seeds from hexagonal crystals of glyIL-2 promotes nucleation of trigonal crystals of unglycosylated IL-2. These trigonal crystals belong to the space group P3,21 or its enantiomorph, with similar cell dimensions to the glyIL-2 hexagonal crystals. Key words: interleukin-2, protein crystallography, glycoprotein INTRODUCTION Interleukin-2 (IL-2) is a lymphokine produced by T lymphocytes which stimulates the growth and differentiation of various subpopulations of lymphocytes (recent reviews','). IL-2-induced activation processes appear to be mediated via an interaction with high-affinity cell surface receptor3 and, as a consequence of this interaction, multiple cell-mediated and humoral immune responses are enhanced. IL-2 is the primary growth factor for the precursors of alloantigen-specific cytotoxic T cells and enhances the proliferation and differentiation of activated B lymphocyte^.^ IL-2 also induces the activation of nonspecific natural killer cells5 and lymphokine-activated killer cells.6 The augmentation of some of the immune responses which have been observed may, in fact, be due to part of the activation of intermediate cells with consequent production of secondary immunostimulatory lymphokines. In this regard, IL-2 has been shown to induce the secretion of 0 1992 WILEY-LISS,
INC.
gamma interferon7 and B cell growth factor' by T lymphocytes which are responsive to interleukin-2. Naturally occurring human IL-2 exhibits a n apparent molecular weight of approximately 17,000. Although considerable isoelectric point heterogeneity exists, due mainly to variable glycosylation including a sialic acid moiety,' it is known that within a given species only a single IL-2 gene codes for the interleukin-2 molecule. The nucleotide sequence for the human protein codes for 153 amino acids of which 20 residues constitute the amino terminal signal peptide.l0-l2 Because of the dramatic immunomodulatory properties of this lymphokine, there has been considerable interest in the evaluation of IL-2 itself, as well as IL-2 antagonists, as potential therapeutic agents in human disorders ranging from cancer to autoimmune disease. Within this context it is not surprising that several groups have reported the crystallization of the Escherichia coli expressed IL-2 (Table I)I3-l6 and have undertaken crystallographic studies toward the elucidation of its structure. A schematic resolution X-ray structure has been published for unglycosylated interleukin216 with crystals that were twinned. The subsequent refinement of the structure has proved problematical (McKay, personal communication). The structure is mainly helical, but the conformations of loops and the positions of side chains remain in doubt. Knowledge of the fine structural details is important in the identification of the functionally important residues and hence in the design of agonists and antagonists. The mammalian expressed material differs from that produced in E . coli because of the O-glycosylation at Thr-3, and in the fact that it is secreted and had not undergone denaturation in guanidinium chloride and subsequent renaturation. The crystallization results reflect these differences, although functionally the recombinant proteins are similar to natural 1L-2.6 By studying the glycosylated IL-2
Received April 16, 1991; accepted April 16, 1991. Address reprint requests to Ian A. Wilson, The Scripps Research Institute, Department of Molecular Biology (MB131, 10666 North Torrey Pines Road, La Jolla, CA 92037.
25
CRYSTALLIZATION STUDIES OF HUMAN rIL-2
TABLE I. Reported Crystal Forms of Human rIL-2
Protein Unglycosylated IL-2 E. coli Cys-Ala 125 Unglycosylated IL-2 E . Coli Unglycosylated IL-2 E. coli Unglycosylated IL-2 E . Coli Glycosylated CHO cells
Space group P1 P2,2,2
Cell dimensions (A) a b c 55.8 40.1 33.7 (Y = 90.0" p = 109.3" y = 93.2" 49.2 87.6 32.4
P6222* P2,2,2 p212121 P3,21* p21
105.8 47.9 48.0 105.0 80.0
P6222*
105.2
105.8 79.6 73.0 105.0 48.0 p = 96.5" 105.2
Maximum resolution
(A, 3.0
Reference Brandhuber et al. (1987)16
2.2
Fujishima et al. (1987)15
122.2 31.9 32.0 122.2 32.0
3.4 2.5 2.5 2.7
Crystallization Studies of Glycosylated and Unglycosylated Human Recombinant Interleukin-2 Enrico A. Stura, Ping Chen, Carrie M. Wilmot, Jairo H. Arevalo, and Ian A. Wilson Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
ABSTRACT Glycosylated interleukin-2 (glyIL-2) has been crystallized in two crystal forms, and unglycosylated interleukin-2 (uIL-2) has been crystallized in three forms. The glycosylated form of the human recombinant IL-2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space group P6,22 or its enantiomorph. The crystals diffract to 2.8 A and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0-5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL-2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.55.6 in the monoclinic space group P2,, and less frequently in the orthorhombic space group P2,2,2, from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross-seeding uIL-2 with seeds from hexagonal crystals of glyIL-2 promotes nucleation of trigonal crystals of unglycosylated IL-2. These trigonal crystals belong to the space group P3,21 or its enantiomorph, with similar cell dimensions to the glyIL-2 hexagonal crystals. Key words: interleukin-2, protein crystallography, glycoprotein INTRODUCTION Interleukin-2 (IL-2) is a lymphokine produced by T lymphocytes which stimulates the growth and differentiation of various subpopulations of lymphocytes (recent reviews','). IL-2-induced activation processes appear to be mediated via an interaction with high-affinity cell surface receptor3 and, as a consequence of this interaction, multiple cell-mediated and humoral immune responses are enhanced. IL-2 is the primary growth factor for the precursors of alloantigen-specific cytotoxic T cells and enhances the proliferation and differentiation of activated B lymphocyte^.^ IL-2 also induces the activation of nonspecific natural killer cells5 and lymphokine-activated killer cells.6 The augmentation of some of the immune responses which have been observed may, in fact, be due to part of the activation of intermediate cells with consequent production of secondary immunostimulatory lymphokines. In this regard, IL-2 has been shown to induce the secretion of 0 1992 WILEY-LISS,
INC.
gamma interferon7 and B cell growth factor' by T lymphocytes which are responsive to interleukin-2. Naturally occurring human IL-2 exhibits a n apparent molecular weight of approximately 17,000. Although considerable isoelectric point heterogeneity exists, due mainly to variable glycosylation including a sialic acid moiety,' it is known that within a given species only a single IL-2 gene codes for the interleukin-2 molecule. The nucleotide sequence for the human protein codes for 153 amino acids of which 20 residues constitute the amino terminal signal peptide.l0-l2 Because of the dramatic immunomodulatory properties of this lymphokine, there has been considerable interest in the evaluation of IL-2 itself, as well as IL-2 antagonists, as potential therapeutic agents in human disorders ranging from cancer to autoimmune disease. Within this context it is not surprising that several groups have reported the crystallization of the Escherichia coli expressed IL-2 (Table I)I3-l6 and have undertaken crystallographic studies toward the elucidation of its structure. A schematic resolution X-ray structure has been published for unglycosylated interleukin216 with crystals that were twinned. The subsequent refinement of the structure has proved problematical (McKay, personal communication). The structure is mainly helical, but the conformations of loops and the positions of side chains remain in doubt. Knowledge of the fine structural details is important in the identification of the functionally important residues and hence in the design of agonists and antagonists. The mammalian expressed material differs from that produced in E . coli because of the O-glycosylation at Thr-3, and in the fact that it is secreted and had not undergone denaturation in guanidinium chloride and subsequent renaturation. The crystallization results reflect these differences, although functionally the recombinant proteins are similar to natural 1L-2.6 By studying the glycosylated IL-2
Received April 16, 1991; accepted April 16, 1991. Address reprint requests to Ian A. Wilson, The Scripps Research Institute, Department of Molecular Biology (MB131, 10666 North Torrey Pines Road, La Jolla, CA 92037.
25
CRYSTALLIZATION STUDIES OF HUMAN rIL-2
TABLE I. Reported Crystal Forms of Human rIL-2
Protein Unglycosylated IL-2 E. coli Cys-Ala 125 Unglycosylated IL-2 E . Coli Unglycosylated IL-2 E. coli Unglycosylated IL-2 E . Coli Glycosylated CHO cells
Space group P1 P2,2,2
Cell dimensions (A) a b c 55.8 40.1 33.7 (Y = 90.0" p = 109.3" y = 93.2" 49.2 87.6 32.4
P6222* P2,2,2 p212121 P3,21* p21
105.8 47.9 48.0 105.0 80.0
P6222*
105.2
105.8 79.6 73.0 105.0 48.0 p = 96.5" 105.2
Maximum resolution
(A, 3.0
Reference Brandhuber et al. (1987)16
2.2
Fujishima et al. (1987)15
122.2 31.9 32.0 122.2 32.0
3.4 2.5 2.5 2.7
