Protein expression and purification from Hi5 insect cells is as described (1, 2).
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1 Materials & methods Protein expression and purification Protein expression and purification from Hi5 insect cells is as described (1, 2). The purified complex exhibited a 2:2:2 stoichiometry as determined by multi-angle light scattering and analytical ultracentrifugation, consistent with previous reports describing the activation complex as a hexamer (3, 4). In order to produce diffraction quality crystals, eight of the ten possible N-linked glycosylation sites were eliminated by mutagenesis, resulting in greatly improved diffraction. To reduce the number of N-linked glycans, 8 mutations were engineered where asparigines were mutated to glutamines (gp130: Asn 21 Asn 109 Asn 135 Asn 205 Asn 224 ; Il-6: Asn 55 Asn 154 ; hil-6rα: Asn 202 ). Asn 61 from gp130 and Asn 202 from IL-6Rα were not mutated. Mutations resulted in reduced yields of the proteins but did not change their biochemical behavior. All proteins used in crystallization were produced from insect cells. Hexa-histidine tags were removed from proteins to be used in crystallization with an overnight digest of carboxypeptidase A (1:100) 4 C. Crystallization, data collection and processing The ternary complex consisting of gp130 D1D2D3, human IL-6 and human IL- 6Rα was purified by superdex200 gel filtration column and further purified by monoq chromatography prior to crystallization. Diamond shaped crystals measuring approximately 0.3 Å in the longest dimension were obtained in 0.5 µl sitting drops with equal volumes of hexamer complex (8 mg/ml) and mother liquor (2.0 M Na formate ph 4.5, 0.1 M sodium acetate ph 4.5). These conditions produced crystals that grew in space
2 group R32 with half of the hexamer in the asymmetric unit of the hexagonal cell (a= 279.8, b= 279.8, c=96.7). Cryo-preservation of the hexamer crystals was obtained in 3 M sodium formate. X-ray diffraction to 3.65 Å was collected at the Lawrence Berkeley National Laboratory on a 2 2 CCD detector with 60 second images. Data was integrated with DENZO and reduced using SCALEPACK (5). Data collection and refinement statistics are presented in Table 1. Structure solution and refinement Initial phases were obtained by molecular replacement with MOLREP (6) using the coordinates of the high resolution crystal structures of human IL-6 structure PDB 1ALU(7) and the D1D2D3 domains of gp130 from the viral IL-6 tetramer PDB 1I1R (2) as search models. The human IL-6Rα model was originally traced de novo into the density using phases derived from IL-6 and gp130, but was later substituted with the high-resolution structure of the IL-6Rα solved by Varghese and coworkers upon release from the PDB (PDB 1N26) (8). The data was refined to a final resolution of 3.65 Å with CNS (9). The model was visualized and built into the electron density map using the program O (10). Several rounds of refinement resulted in an overall R cryst of 24.8% and an R free of 31.7%. The final model of LIF begins at residue Cys 12 and ends at Phe 180 while the gp130 model incorporates residues Gly 101 to Glu 301. Stereochemical analysis was performed with PROCHECK (11) with the Ramachandran plot showing no nonglycine residues modeled in disallowed orientations.
3 Isothermal titration calorimetry We expressed the minimal gp130-d2d3 to trap the complex in the intermediate site II recognition complex. We expressed the D1D2D3 domain construct used in the crystal structure to characterize the both site II and III interfaces within the contest of the hexamer, and finally a D1D2D3D4D5D6 full-length soluble version of gp130 to assess the affect of the D4D5D6 membrane proximal domains on hexamer formation. For IL-6, in order to eliminate the simultaneous binding equilibrium of the IL-6/IL-6Rα binary complex during trimolecular titrations with gp130, we engineered a single-chain complex of IL-6/IL-6Rα, which cannot dissociate and so represents a fixed site II surface. The single-chain version of the IL-6/IL-6Rα complex was utilized for these measurements in order to deconvolute the trimolecular equilibrium (i.e. IL-6 + IL-6Rα + gp130) into a bimolecular interaction event (i.e. IL-6/IL-6Rα + gp130). A fifteen amino acid Gly-Ser linker extending from the C-terminus of the receptor to the N-terminus of IL-6 covalently links the active binary complex. In this way, the titration of the IL-6/IL-6Rα complex into gp130 does not represent a complicated association/dissociation equilibrium resulting from both IL-6/IL-6Rα and IL-6/IL-6Rα/gp130 interactions. We have previously shown that this single-chain complex behaves in all respects like the untethered binary complex with respect to biological activity and interaction with gp130 (4, 12) (data not shown). Calorimetric titrations were carried out on a VP-ITC calorimeter (MicroCal, Northhampton, MA). Prior to each titration, the protein samples were degassed for 10 minutes. Data was processed with the MicroCal Origin 5.0 software. The same buffer of 10 mm HEPES ph 7.5 supplemented with 200 mm sodium chloride was used in each
4 experiment to control for buffer heat dilution effects. The binding site molar concentrations of the samples in the sample cell were in the range of µm. The equivalent binding site molar concentrations in the injection syringe was at least seven fold greater than that of the cell. Multi angle light scattering A DAWN EOS (Wyatt Technology, Santa Barbara, CA) equipped with a K5 flow cell and a 30 mw linearly polarized GaAs laser of wavelength 690 nm was used in all experiments. All measurements were made in the in-line flow mode. A Jasco Model PU- 980 (Jasco Corp, Tokyo, Japan) pump was used to flow 0.1 µm filtered solvent (10 mm HEPES ph mm NaCl) through a Shimadzu DGU-14A (Shimadzu Corp., Kyoto, Japan) degasser and into an HR 10/30 Superdex-200 (Amersham Biosciences, Piscataway, NJ) gel filtration column. The sample was at approximately 1.5 mg/ml (in the eluent buffer). Both the light scattering unit and the refractometer were calibrated as per the manufacturer s instructions. A value of ml/g was assumed for the dn/dc of the protein. Light scattering data was used from 11 detectors ranging from 50.0 o to o (detectors 6 through 16). The detector responses were normalized by measuring the signal from monomeric bovine serum albumin. The temperature of the light scattering unit was maintained at 25 o C and the temperature of the refractometer was maintained at 35 o C. The column and all external connections were at ambient temperature (20-22 o C). The flow rate was maintained at 0.5 ml/minute throughout the experiments.
5 REFERENCES 1. X. He, D. Chow, M. M. Martick, K. C. Garcia, Science 293, 1657 (2001). 2. D. Chow, X. He, A. L. Snow, S. Rose-John, K. C. Garcia, Science 291, 2150 (2001). 3. L. D. Ward et al., J Biol Chem 269, (1994). 4. D. Chow, J. Ho, T. L. Nguyen Pham, S. Rose-John, K. C. Garcia, Biochemistry 40, 7593 (2001). 5. Z. Otwinowski, W. Minor, Methods Enzymol. 276, 307 (1997). 6. V. Vagin, A. Teplyakov, J. Appl. Cryst. 30, 1022 (1997). 7. W. Somers, M. Stahl, J. S. Seehra, Embo J 16, 989 (1997). 8. J. N. Varghese et al., Proc Natl Acad Sci U S A 99, (2002). 9. A. T. Brunger et al., Acta. Cryst. D54, 905 (1998). 10. T. A. Jones, J. Y. Zhou, S. W. Cowan, M. Kjeldgaard, Acta. Cryst. A47, 110 (1991). 11. R. A. Laskowksi, M. W. MacArthur, D. S. Moss, J. M. Thornton, J. Appl. Cryst 26, 283 (1993). 12. M. Fischer et al., Nat Biotechnol 15, 142 (1997).
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