title: Fast binding kinetics and conserved 3D structure underlie the antagonistic activity of mutant TNF: useful information for designing artificial proteo-antagonists.
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.
authors.affiliation: Osaka University, Yamadaoka, Suita, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Mukai
authors.forename: Teruya
authors.initials: T
authors.lastname: Nakamura
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yoshioka
authors.forename: Hiroko
authors.initials: H
authors.lastname: Shibata
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Abe
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nomura
authors.forename: Madoka
authors.initials: M
authors.lastname: Taniai
authors.forename: Tsunetaka
authors.initials: T
authors.lastname: Ohta
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tsunoda
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Kamada
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Yamagata
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology
chemical_list: D014409:Tumor Necrosis Factor-alpha