Crystal structure determination TNF

The soluble form of human TNF (CID2043, UniProt {"type":"entrez-protein","attrs":{"text":"P01375","term_id":"135934","term_text":"P01375"}}P01375, residues 77-233) was generated as described above. The product contained an N-terminal serine residue as a cloning artefact but this was not part of the native sequence of the TNF molecule. The extracellular domain of human TNFR1 (CID5602, UniProt {"type":"entrez-protein","attrs":{"text":"P19438","term_id":"135959","term_text":"P19438"}}P19438, residues 42-184) with N54D and C182S mutations was expressed as a secreted protein in baculovirus-infected Trichoplusia ni insect cells (Expression Systems, catalogue number 94-002 F and 91-002) using ESF-921 insect cell media (Expression Systems, catalogue number 96-001-01). An extra glycine was present at the N-terminus of the protein due to a cloning artifact. This is not part of the native sequence of the TNFR1. The fusion protein plasmid was cloned into the pEMB50 expression vector, which encodes a cleavable N-terminal secretion signal and His-tagged fusion protein. Virus was generated using the baculovirus expression system. Infected insect cells secreted the fusion protein into the media. The fusion protein was purified by Ni-NTA chelate chromatography and eluted from the Ni column using an imidazole gradient. The eluted protein was cleaved with protease to release the N-terminal His-fusion tag. The cleaved TNFR1 was subsequently purified by a subtractive Ni chelate chromatography step and further purified by size exclusion chromatography. The final TNFR1 product was typically concentrated to 10.0 mg/ml and flash-frozen in liquid nitrogen.

To form the complex, 333 µl of purified human TNF at 300 mM was mixed with 4567 µl of SEC buffer (10 mM HEPES, pH 7.5, 150 mM NaCl) and 100 µL of UCB-8733 (10 mM in DMSO, approximately 10 molar excess) and incubated at 4 °C overnight. The following day, the complex was formed by adding 4080 µl of SEC buffer to 700 µl TNFR1, 5,000 µl of TNF/UCB-8733 mix, and 220 µl CA1974 Fab fragment at 500 mM. Total volume of the reaction was 10 ml with a final molar ratio of 3 TNF monomers (equivalent to 1 trimer): 2.5 TNFR1 receptors: 1.2 Fab. The ternary complex (cytokine, small molecule inhibitor, receptor, and Fab) was incubated for 1 h at 4 °C. Analytical SEC was used to assess complex formation (Supplementary Fig. ²). The sample was then concentrated to 1.5 ml and was loaded in a single injection on Superdex 200 16/600 size exclusion column (120 ml) pre-equilibrated with 10 mM HEPES pH 7.5, 150 mM NaCl. Peak fractions of the ternary complex were selected and concentrated to 13.7 mg/ml and immediately used in crystallization trials. The ternary complex was crystallized by sitting drop vapour diffusion by mixing 0.5 μl of complex with 0.5 μl of Wizard III/IV: 0.1 M HEPES, pH7.0, 10% PEG6,000 (condition B8) over 80 μl of the same crystallization solution. Crystals were harvested for data collection approximately 2 months after initial set-up. They were cryo-protected in glycerol performed in 5-10-15% steps, then frozen directly in liquid nitrogen for data collection at Advanced Photon Source at Argonne National Laboratory, Life Sciences Collaborative Access Team (LS-CAT), beamline 21-ID-F (wavelength 0.9786 Å, 100 K).

Crystallization of TNF with UCB-8733 was achieved using methods previously described⁵. In brief, TNF at 4-7 mg/ml was incubated with 0.5 mM UCB-8733 overnight at 4 °C. Crystals were grown at 16 °C by sitting drop vapour diffusion with a mixture of 0.5 µl protein:compound and 0.5 µl of 24.4% w/v PEG 3350, 0.1 M HEPES, pH 7.0. Crystals were cryo-protected in 10% ethylene glycol and frozen in liquid nitrogen for data collection. The data set was collected at APS, LS-CAT, beamline 21-ID-G (wavelength 0.97872 Å, 100 K). The structure of the human TNF (CID2043), human TNFR1 (CID5602), and CA1974 Fab complex with small-molecule UCB-8733 was solved by molecular replacement using Phenix.Phaser²⁹ with input models based upon previously determined unpublished structures (Supplementary Table ³). Data were integrated in XDS and scaled using XSCALE³⁰. Initial structure determination and refinement used data to 3.00 Å resolution from a single crystal. Iterative manual model building using Coot³¹ and in Phenix.Refine²⁹ continued until R and R_free reached R = 0.201, R_free = 0.259 (Ramachandran favoured = 97.55%, outliers = 0.25%). Diffraction data for TNF and small-molecule UCB-8733 were reduced and scaled as above and the structure was solved by rigid body refinement in Refmac³² using a previously determined structure as the input model. Model building was performed using iterative rounds of Coot and Refmac until R and R_free converged at 0.184/0.270, respectively (Ramachandran favoured = 97.24%, outliers = 0.09%). Model quality was validated using Coot and MolProbity prior to deposition in the Protein Data Bank (PDB codes 7KPB/7KPA)^33–35. Final data processing and refinement statistics are listed in Supplementary Table ³. Electron density images are shown in Supplementary Fig. ³ (human TNF [bound with UCB-8733) in complex with human TNFR1 and CA1974 Fab) and 6 (human TNF bound with UCB-8733).