PDBsum entry 2e7a

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Cytokine PDB id
Protein chains
150 a.a.
142 a.a.
Waters ×237

References listed in PDB file
Key reference
Title Creation and X-Ray structure analysis of the tumor necrosis factor receptor-1-Selective mutant of a tumor necrosis factor-Alpha antagonist.
Authors H.Shibata, Y.Yoshioka, A.Ohkawa, K.Minowa, Y.Mukai, Y.Abe, M.Taniai, T.Nomura, H.Kayamuro, H.Nabeshi, T.Sugita, S.Imai, K.Nagano, T.Yoshikawa, T.Fujita, S.Nakagawa, A.Yamamoto, T.Ohta, T.Hayakawa, T.Mayumi, P.Vandenabeele, B.B.Aggarwal, T.Nakamura, Y.Yamagata, S.Tsunoda, H.Kamada, Y.Tsutsumi.
Ref. J Biol Chem, 2008, 283, 998. [DOI no: 10.1074/jbc.M707933200]
PubMed id 18003610
Tumor necrosis factor-alpha (TNF) induces inflammatory response predominantly through the TNF receptor-1 (TNFR1). Thus, blocking the binding of TNF to TNFR1 is an important strategy for the treatment of many inflammatory diseases, such as hepatitis and rheumatoid arthritis. In this study, we identified a TNFR1-selective antagonistic mutant TNF from a phage library displaying structural human TNF variants in which each one of the six amino acid residues at the receptor-binding site (amino acids at positions 84-89) was replaced with other amino acids. Consequently, a TNFR1-selective antagonistic mutant TNF (R1antTNF), containing mutations A84S, V85T, S86T, Y87H, Q88N, and T89Q, was isolated from the library. The R1antTNF did not activate TNFR1-mediated responses, although its affinity for the TNFR1 was almost similar to that of the human wild-type TNF (wtTNF). Additionally, the R1antTNF neutralized the TNFR1-mediated bioactivity of wtTNF without influencing its TNFR2-mediated bioactivity and inhibited hepatic injury in an experimental hepatitis model. To understand the mechanism underlying the antagonistic activity of R1antTNF, we analyzed this mutant using the surface plasmon resonance spectroscopy and x-ray crystallography. Kinetic association/dissociation parameters of the R1antTNF were higher than those of the wtTNF, indicating very fast bond dissociation. Furthermore, x-ray crystallographic analysis of R1antTNF suggested that the mutation Y87H changed the binding mode from the hydrophobic to the electrostatic interaction, which may be one of the reasons why R1antTNF behaved as an antagonist. Our studies demonstrate the feasibility of generating TNF receptor subtype-specific antagonist by extensive substitution of amino acids of the wild-type ligand protein.
Figure 6.
FIGURE 6. Overall structures of R1antTNF and wtTNF. A, refined structure of the R1antTNF trimer (green). Blue spheres show the mutated residues(amino acids 84-89) in R1antTNF. This structure is registered in the PDB (PDB code 2E7A). B, structure of the wtTNF trimer (gray). This structure has been published, and its PDB code is 1TNF. C, model structures of the TNF-TNFR1 complexes. Each TNF is superposed on the LT- derived from the LT- -TNFR1 complex (PDB code 1TNR). TNF binds to three R1 monomers on the cell surface. TNFR1s are shown using red schematics. Superposition of the structures of the wtTNF and R1antTNF was performed using the ccp4i program.
Figure 7.
FIGURE 7. Structural difference between the receptor binding region of the R1antTNF and wtTNF. A, interaction between the wtTNF (gray) and TNFR1 (red). White layer depicts the molecular surface of the TNFR1. Hydrophobic interaction is formed between the Tyr-87 and molecular pocket in the TNFR1 (Leu-67, Leu-71, Ala-62, and Ser-63). B, interaction between the R1antTNF (green) and TNFR1 (red). Yellow broken lines show the possible interactions of the R1antTNF His-87 with the receptor Ser-63 and Glu-64. In this simulation, the side chains of each structure were rotated to fit the predicted interaction. Stable structures of these rotamers were constructed using the O program.
The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 998) copyright 2008.
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