Structural Preparation of TNF-α–TNFR1 Complex and TNF-α.

To date, no NMR/X-ray crystal structures are available for the human TNF-α–TNFR1 complex. However, several studies have reported the structures of the TNF-α–TNFR2 complex and the extracellular domain of TNFR1 determined by single-crystal X-ray diffraction.^21,22 The TNF-α–TNFR1 complex was obtained on the basis of the crystal structures of TNFR1 (PDB entry 1EXT, resolution 1.85 Å) and TNF-α–TNFR2 (PDB code 3ALQ, resolution 3 Å). The relative orientations of TNF-α and TNFR1 were based on the crystal structure of TNF-α bound with TNFR2. Superimposition and adjustment were applied using PyMOL (www.pymol.org) to obtain a TNF-α–TNFR1 complex. Briefly, TNFR1 (sequence ID {"type":"entrez-protein","attrs":{"text":"P19438","term_id":"135959","term_text":"P19438"}}P19438–1) was superimposed onto the α-carbon (Cα) atoms of TNFR2 (especially for M³⁰, H⁴⁰, C⁹⁶, C¹¹⁵, and G¹²⁶; sequence ID {"type":"entrez-protein","attrs":{"text":"P20333","term_id":"21264534","term_text":"P20333"}}P20333–1). In addition, the crystal structure of human TNF-α with the antagonist SPD-304 (PDB code 2AZ5, resolution 2.1 Å) was used. The structures were downloaded from the PDB (http://www.rcsb.org/). SYBYL-X 1.3²³ was used to prepare the structure, including residual repair and energy minimization. The detailed parameters were described in our previous publications.^23,25 ProSA-web Z-scores²⁶ and ProCheck Ramachandran plots²⁷ were used for structural stereochemical evaluation of the TNF-α–TNFR1 complex. The protein structure of the TNF-α–TNFR1 complex can be downloaded from our public Web site (http://www.cbligand.org/downloads/TNF_TNFR1.pdb).