Creation and X-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-alpha antagonist.

Abstract

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.

• Authors: Hiroko Shibata, Yasuo Yoshioka, Akiko Ohkawa, Kyoko Minowa, Yohei Mukai, Yasuhiro Abe, Madoka Taniai, Tetsuya Nomura, Hiroyuki Kayamuro, Hiromi Nabeshi, Toshiki Sugita, Sunao Imai, Kazuya Nagano, Tomoaki Yoshikawa, Takuya Fujita, Shinsaku Nakagawa, Akira Yamamoto, Tsunetaka Ohta, Takao Hayakawa, Tadanori Mayumi, Peter Vandenabeele, Bharat B Aggarwal, Teruya Nakamura, Yuriko Yamagata, Shin-ichi Tsunoda, Haruhiko Kamada, Yasuo Tsutsumi
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 283 Issue 2 Pg. 998-1007 (Jan 11 2008) ISSN: 0021-9258 [Print] United States
• PMID: 18003610 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Receptors, Tumor Necrosis Factor, Type I
• Tumor Necrosis Factor-alpha

Topics

• Amino Acid Sequence
• Animals
• Base Sequence
• Binding Sites
• Crystallography, X-Ray
• Humans
• Kinetics
• L Cells
• Mice
• Models, Molecular
• Protein Conformation
• Receptors, Tumor Necrosis Factor, Type I (chemistry, drug effects, genetics)
• Reverse Transcriptase Polymerase Chain Reaction
• Tumor Necrosis Factor-alpha (antagonists & inhibitors, genetics)