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Journal of Biochemistry Advance Access originally published online on April 22, 2009
Journal of Biochemistry 2009 146(2):167-172; doi:10.1093/jb/mvp065
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Fast Binding Kinetics and Conserved 3D Structure Underlie the Antagonistic Activity of Mutant TNF: Useful Information for Designing Artificial Proteo-Antagonists

Yohei Mukai1,2, Teruya Nakamura3, Yasuo Yoshioka2,4, Hiroko Shibata2, Yasuhiro Abe2, Tetsuya Nomura1,2, Madoka Taniai5, Tsunetaka Ohta5, Shinsaku Nakagawa1, Shin-ichi Tsunoda2, Haruhiko Kamada2, Yuriko Yamagata3 and Yasuo Tsutsumi1,2,*

1Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871; 2Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Osaka 567-0085; 3Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973; 4The Center for Advanced Medical Engineering and Informatics, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871; and 5Hayashibara Biochemical Laboratories, Inc., 1-2-3 Shimoishii, Okayama 702-8006, Japan

*To whom correspondence should be addressed. Tel: +81-6-6879-8230, Fax: +81-6-6879-8234, E-mail: ytsutsumi{at}phs.osaka-u.ac.jp

Received February 18, 2009; Accepted March 17, 2009


  Abstract

Tumour necrosis factor (TNF) is an important cytokine that induces an inflammatory response predominantly through the TNF receptor-1 (TNFR1). A crucial strategy for the treatment of many autoimmune diseases, therefore, is to block the binding of TNF to TNFR1. We previously identified a TNFR1-selective antagonistic mutant TNF (R1antTNF) from a phage library containing six randomized amino acid residues at the receptor-binding site (amino acids 84–89). Two R1antTNFs, R1antTNF-T2 (A84S, V85T, S86T, Y87H, Q88N and T89Q) and R1antTNF-T8 (A84T, V85P, S86A, Y87I, Q88N and T89R), were successfully isolated from this library. Here, we analysed R1antTNF-T8 using surface plasmon resonance spectroscopy and X-ray crystallography to determine the mechanism underlying the antagonistic activity of R1antTNF. The kinetic association/dissociation parameters of R1antTNF-T8 were higher than those of wild-type TNF, indicating more rapid bond dissociation. X-ray crystallographic analysis suggested that the binding mode of the T89R mutation changed from a hydrophobic to an electrostatic interaction, which may be responsible for the antagonistic behaviour of R1antTNF. Knowledge of these structure–function relationships will facilitate the design of novel TNF inhibitors based on the cytokine structure.

Key Words: antagonistic activity, mutant, structure–function relationship, tumour necrosis factor (TNF), X-ray crystallography


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