www.sciencemag.org/cgi/content/full/310/5751/1159/dc1 Supporting Online Material for Structure of the Quaternary Complex of Interleukin-2 with Its α, β, and γ c Receptors Xinquan Wang, Mathias Rickert, K. Christopher Garcia* *To whom correspondence should be addressed. E-mail: kcgarcia@stanford.edu Published 18 November 2005, Science 310, 1159 (2005) DOI: 10.1126/science.1117893 This PDF file includes: Materials and Methods Figs. S1 to S3 Tables S1 and S2 References
Supporting Online Material Materials and Methods Protein expression and purification All proteins used in this study were expressed using the Baculovirus system (Pharmingen) in insect cells as described (1). Briefly, Insect Spodoptera frugiperda (Sf9) cells were used for generating high titer recombinant virus and were cultured at 28 C using SF900 II SFM medium (Invitrogen). Trichopulsia ni (High-Five ) cells (Invitrogen) were used to express the recombinant protein and were grown in Insect Xpress medium (Cambrex) at 28 C (2, 3). Full length IL-2 (residues 1-133), the ectodomains of IL-2Rα (residue 1-217), IL-2Rβ (residue 1-214), and N-terminal deleted γ c (residue 34-232) were cloned with C-terminal hexa-histidine tag into the pacgp67a vector (Pharmingen). After infection of High-Five cells with recombinant virus, the proteins were purified by Ni-NTA and concentrated using Centricon (Millipore, Bedford, MA) spin concentrators and purified with an FPLC Superdex 200 sizing column (Pharmacia, NJ). To improve crystal quality and diffraction power, N-linked glycosylation sites at IL-2Rα residue Asn 68, IL-2Rβ residues Asn 3, 17, 45 and γ c residues Asn 53 were mutated to Glutamine residues. Previous studies to determine the binding epitope between γ c and IL- 4 showed that the first 33 N-terminal residues, Leu 1 -Leu 33, of γ c can be deleted without loosing any binding reactivity to IL-4 (4). To remove the potential O-linked glycosylation sites within the N-terminus of γ c, we inserted the same N-terminal deletion of γ c as described by Zhang et al., 2002 (4), without loosing binding reactivity to IL-2 (results not shown). In addition to the inserted mutations within the γ c molecule we produced γ c in 1
Hi-5 insect cells in the presence of 0.1 mm Swainsonine (Biomol, PA, USA), an inhibitor of lysosomal alpha-mannosidase and of the Golgi complex alpha-mannosidase II (5, 6). Incubation with the alkaloid Swainsonine lead to a decrease in non-homogeneously long complex type oligosaccharides and to an increase in more homogenously long mannose type oligosaccharides which could be digested with endoglucosaminiase H (Endo H, New England Biolabs, Beverly, USA, 20000 U/mg glycopeptide) (5). Wild-type IL-2Rα was reduced with 10mM Cysteine (Sigma) and alkylated with 20mM Iodoacetamide, to prevent disulfide formation by the free cysteine, Cys 192. As previously reported the alkylation, or removal of Cys 192 has no influence on ligand binding (7, 8). Crystallization, data collection and processing The quaternary complex consisting of IL-2, IL-2Rα, IL-2Rβ and N-terminal deleted γ c was purified by Superdex 200 gel filtration column and further purified by MonoQ chromatography. Hexa-histidine tags were removed form proteins with an overnight digest of carboxypeptidase A plus B (1:100) at 4 C. Prior to crystallization carboxypeptidase A plus B was removed by gel filtration in HBS buffer (10 mm Hepes (ph 7.5) supplemented with 150 mm sodium chloride) and the protein complex was concentrated to 6 7 mg/ml. The crystals were obtained through vapor-diffusion in 0.5 µl sitting drops with equal volumes of protein complex and mother liquor (29% Pentaerythritol Ethoxylate 15/4; 50 mm Ammonium Sulfate; 50 mm Bis Tris, ph 6.1). Data sets were measured on a 2 x 2 CCD array (ADSC) at Berkeley Advanced Light Source (ALS, beam line 8.3.1). Crystals were cryoprotected before cooling to 100K 2
with 5% Glycerol in the mother liquor. The native data set was collected to 2.3 Å. The crystals are space group C2, with cell dimensions a = 113.9 Å, b = 87.7 Å, c = 130.2 Å. The data were indexed, integrated and scaled with HKL2000 (9). Structure solution and refinement The quaternary complex structure was determined by molecular replacement method using data between 20.0 and 30 Å with the program Phaser (10). The first search model being tried is the binary complex structure of IL-2/IL-2Rα (PDB ID: 1Z92), but no molecular replacement solution was found. Using IL-2 only from the binary complex as search model, the top solution from Phaser has a Z-score of 7.62. After fixing the solution of IL-2, we tried to determine the position of the second molecule (IL-2Rβ or γ c ) by searching the known structures in the class I cytokine receptor family. Two significant solutions came out from the screening: one has a Z-score of 10.86 with the structure of IL-4 alpha receptor (PDB ID: 1IAR) as search model, the other has a Z-score of 10.40 with the structure of erythropoietin receptor (PDB ID: 1ERN) as search model. Checking both solutions on the graphics revealed that these two models were positioned in different sides of IL-2 to form a compact tri-molecular complex. After rigid-body refinement, the R and R free factors were 48.5% and 48.3%, respectively, in the resolution range of 20-2.3 Å. The tri-molecular model consisting of IL-2, IL-2Rβ and γ c had R and R free factors of 33.2% and 37.7%, respectively, after rounds of model building followed by simulated annealing, positional, and individual B factor refinements. The electron density map at this point clearly showed the region of IL-2Rα. The D1 domain of IL-2Rα responsible for the binding with IL-2 could be put into the density without much effort, but the 3
positional shift and flexibility made it necessary to retrace the D2 domain of IL-2Rα. The TLS (11) refinement was introduced in the last step, and the final R and R free factors of the quaternary complex model are 22.3% and 26.9%, respectively. Program CNS (12) and Refmac5 (11) were used for the structural refinement, and model building was carried out using the program O (13). 4
Fig. S1. Open book surface representations of the binding interfaces within the quaternary signaling complex. Each binding area is shown in the color of the contacted cytokine or receptors. Program PyMol (14) was used to make figs. 1 and 2. 5
Fig. S2. (A) The representative electron density map (σ A -weighted 2Fo-Fc) around residues in the IL-2/IL-2Rβ interface. (B) Electron density map (σ A -weighted 2Fo-Fc) around residues in the IL-2/γ c interface. Fig. S3. Sequence alignment of γ c -dependent cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Residues in IL-2 having contact with IL-2Rα, IL-2Rβ, and γ c were labeled with α (cyan), β (blue), and γ (orange). The alignment was done with program ClustalW (15) and figure was prepared with program ESPript (16). 6
Table S1: Crystallographic statistics Data collection Space group C2 Cell demensions a (Å) 113.9 b (Å) 87.7 c (Å) 130.2 β ( ) 116.3 Resolution (high resolution shell) (Å) 50.0-2.30 (2.38-2.30) Completeness (%) 97.4 (85.3) R merge 0.049 (0.425) Refinement statistics Resolution (high resolution shell) (Å) 50.0-2.30 (2.36-2.30) R cryst 0.223 (0.320) R free 0.269 (0.376) r.m.s.d. bond length (Å) 0.012 r.m.s.d. bond angle, dihedral, improper ( ) 1.3, 27.0, 1.5 Average B-factors (Å 2 ) protein 57.7 carbohydrate 63.2 water 55.5 Ramachandran plot (favored, allowed, generous, disallowed ) (%) 86.3, 12.6, 1.1, 0.0 7
Table S2_a. interaction between IL-2 and IL-2Rα Hydrogen bonds and salt bridges IL-2 IL-2Rα Distance (Å) Arg38 NH1 Asp6 OD1 3.2 Arg38 NH2 Cys3 O 3.3 Arg38 NH2 Asp4 O 2.8 Arg38 NH2 Asp5 O 2.8 Arg43 NZ Glu29 OE2 2.6 Tyr45 OH Arg36 N 3.2 Glu61 O Ser39 N 3.0 Glu61 O Ser39 OG 3.3 Glu62 OE1 Arg36 NH1 3.4 Glu62 OE2 Arg36 NH2 2.7 Glu68 OE1 Leu42 N 3.0 Glu68 OE2 Ser41 OG 3.2 Glu68 O Tyr43 OH 3.2 Vdw contacts IL-2 IL-2Rα Lys35 Leu2 Arg38 Cys3, Asp4, Asp5, Asp6, His120 Phe42 Asn27, Leu42, Tyr43, His120 Lys43 Glu29, Arg36, Leu42 Phe44 Leu42 Tyr45 Arg35, Arg36 Glu61 Lys38, Ser39 Glu62 Arg36 Lys64 Ser39, Ser41 Pro65 Arg36, Gly40, Leu42 Glu68 Ser41, Leu42, Tyr43 Val69 Leu42 Leu72 Met25, Tyr43 Tyr107 Arg35 8
Table S2_b. interaction between IL-2 and IL-2Rβ Hydrogen bonds and salt bridges IL-2 IL-2Rβ Distance (Å) Glu15 OE2 His138 NE2 2.6 Asp20 OD1 His133 NE2 2.8 Asp20 OD2 Tyr134 OH 2.3 Asp84 OD2 Lys71 NZ 2.9 Ser87 OG Arg42 NH1 2.9 Asn88 OD1 Arg42 NH2 3.0 Asn88 ND2 Gln70 O 3.0 Glu95 OE1 Arg41 NH1 3.2 Hydrogen bonds through water molecules wat-2 O His16 (IL-2) ND1 2.9 wat-2 O Thr74 (IL-2Rβ) N 3.0 wat-24 O His16 (IL-2) NE2 2.9 wat-24 O Tyr134 (IL-2Rβ) O 2.5 wat-25 O Asp84 (IL-2) OD1 2.7 wat-25 O Gln70 (IL-2Rβ) N 2.8 wat-50 O Asp84 (IL-2) O 3.4 wat-50 O Ser87 (IL-2) OG 3.2 wat-50 O Asn88 (IL-2) OD1 2.5 wat-50 O Gln70 (IL-2Rβ) O 2.7 wat-50 O wat-132 O 3.1 wat-63 O Val91 (IL-2) O 2.4 wat-63 O Arg41 (IL-2Rβ) NH1 3.0 wat-113 O Asp20 (IL-2) OD1 3.0 wat-113 O Arg81 (IL-2) NH1 3.2 wat-113 O Asn88 (IL-22) ND2 2.8 wat-113 O Tyr134 (IL-2Rβ) OH 3.2 wat-132 O Trp44 (IL-2Rβ) NE1 3.0 wat-132 O Lys71 (IL-2Rβ) O 2.6 Vdw contacts IL-2 IL-2Rβ Leu12 Gln188 Gln13 Thr74, Val75 Glu15 His138 His16 Thr74, Tyr134, Gln188 Leu19 His133, Tyr134 Asp20 His133, Tyr134 9
Met23 Arg81 Asp84 Ser87 Asn88 Val91 ILe92 Glu95 His133 Arg15,Gln70, His133 Ser69, Gln70, Lys71 Arg42 Arg42, Gln70, Thr73, Tyr134 Arg41, Arg42, Thr73, Val75 Thr73, Tyr34 Arg41, Val75 10
Table S2_c. interaction between IL-2 and γ c Hydrogen bonds and salt bridges IL-2 γ c Distance (Å) Gln22 NE2 Pro207 O 3.3 Thr123 OG1 Gln127 OE1 3.1 Gln126 NE2 Pro207 O 3.1 Gln126 NE2 Ser211 OG 3.1 Ile129 O His159 NE2 3.0 Hydrogen bonds through water molecules wat-129 O Gln126 (IL-2) OE1 2.8 wat-129 O Gln127 (γ c ) OE1 3.3 wat-129 O Asn128 (γ c ) OD1 2.9 Vdw contacts IL-2 Gln11 Glu15 Leu18 Gln22 Glu110 Asn119 Thr123 Gln126 Ser127 Ile129 Ser130 Thr133 γ c His159 Leu208 Pro207, Leu208 Pro207, Ser211 Asn71 Lys125 Tyr103, Gln127 Tyr103, Gln127, Asn206, Pro207, Leu208, Cys209, Gly210, Ser211 Tyr103 His159, Leu208 Tyr103, His159, Cys209 His159 11
Table S2_d. interaction between IL-2Rβ and γ c Hydrogen bonds and salt bridges IL-2Rβ γ c Distance (Å) Arg137 NH1 Ser179 OG 2.6 Arg137 NH1 Glu162 OE1 2.9 Arg137 NH2 Glu162 OE1 2.8 Arg137 O Arg183 NH1 3.0 His138 ND1 Asp181 OD1 2.7 Leu139 O Arg183 NH2 2.5 Leu157 O Gln147 NE2 2.9 Thr159 N Gln147 OE1 3.3 Lys161 N Ser187 OG 2.8 Lys161 NZ Glu149 OE2 2.7 Gln162 OE1 Ser187 N 2.8 Gln162 NE2 Ser187 O 3.0 Lys163 O Gln178 NE2 2.7 Glu170 N Ser190 OG 2.8 Hydrogen bonds through water molecules wat-3 O Gln164 (IL-2Rβ) NE2 2.9 wat-3 O Gln164 (IL-2Rβ) OE1 3.0 wat-3 O Ser187 (γ c ) O 2.6 wat-19 O Lys161 (IL-2Rβ) O 2.9 wat-19 O Lys185 (γ c ) O 2.8 wat-19 O Arg183 (γ c ) O 3.2 wat-92 O Thr159 (IL-2Rβ) O 2.9 wat-92 O Gln147 (γ c ) OE1 2.7 wat-165 O Arg137 (IL-2Rβ) NH1 2.9 wat-165 O His163 (γ c ) O 2.9 wat-165 O Ser179 (γ c ) OG 2.4 Vdw contacts IL-2Rβ Arg121 Glu136 Arg137 His138 Leu139 Glu140 Leu157 Leu158 γ c Lys195 Pro207 Glu162, Ser179, Val180, Asp181, Arg183, Pro207 Asp181, Tyr182, Arg183 Arg183 Arg183 Gln147 Gln147, Pro189 12
Thr159 Leu160 Lys161 Gln162 Lys163 Gln164 Trp166 Ile167 Cys168 Leu169 Glu170 Thr171 Leu187 NAG100 Ser187, Pro189 Ser187, Pro189 Glu149, Arg183, Lys185, Ser187 Phe186, Ser187 Gln178 Gln178, Phe186 Tyr167, Thr176 Pro189 Ser190 Ser190 Ser190, Lys195 Val191 Arg183 Thr176 13
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