Mapping of receptor binding sites on IL-1β by reconstruction of IL-1ra- like domains

D. Boraschi, P. Bossu, P. Ruggiero, A. Tagliabue, R. Bertini, G. Macchia, C. Gasbarro, L. Pellegrini, G. Melillo, E. Ulisse, U. Visconti, C. Bizzarri, E. Del Grosso, A. R. Mackay, G. Frascotti, F. Frigerio, R. Grifantini, G. Grandi

Research output: Contribution to journalArticlepeer-review

Abstract

Upon structure comparison between IL-1β and its antagonist IL-1ra, single or multiple residues along the IL-1β sequence were replaced with the corresponding amino acids present in the IL-1ra protein, in the attempt to identify sites important for receptor binding and for biologic activity on the two molecules. Ten of fifteen mutant proteins had activity comparable to that of wild-type IL-1β in three different biologic assays and in receptor binding, indicating that the introduced changes did not influence the functional structure of the protein. Conversely, three mutants (SMIL- 9:127/263 R/T→W/Y; SMIL-10: 125/127/263/265 T/R/T/Q→R/W/Y/E; SMIL- 15:222/227 I/E→S/S) showed an increased binding capacity for IL-1R(I), not paralleled by increased agonist activity, indicating that the introduced IL- 1ra residues could be involved in the nonagonist IL-1R(I) binding site. On the other hand, two mutants showed diminished binding capacity with concomitant decrease in biologic activity. Both mutants (SMIL-1, five substitutions in the loop 202-214; and SMIL-3, total replacement of the loop 164-173 with the IL-1ra stretch 52-55) included substitutions of residues allegedly important for agonist binding to IL-1R(I). Mutant SMIL-3 showed the most profound reduction in binding capacity for IL-1R(I) (CDw121a) and a more than 1,000-fold reduced biologic activity both in vitro and in vivo, but it retained full capacity of binding to IL-1R(II) (CDw121b) and acted as a selective antagonist of IL-1R(II). From these results the following conclusions can be drawn. IL-1β binds to IL-1R(I), and to IL-1R(II) through different sites, and the loop 164-173 appears as one of the areas involved in the selective interaction with IL-1R(I). Agonist (IL-1β) and nonagonist (IL- 1ra) binding to IL-1R(I) occur through distinct sites, with loops 164-173 and 202-214 of IL-1β identified as two of the sites selectively involved in agonist binding to the activating receptor.

Original languageEnglish
Pages (from-to)4719-4725
Number of pages7
JournalJournal of Immunology
Volume155
Issue number10
Publication statusPublished - 1995

ASJC Scopus subject areas

  • Immunology

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