MAPPING OF RECEPTOR-BINDING SITES ON IL-1-BETA BY RECONSTRUCTION OF IL-1RA-LIKE DOMAINS

Upon structure comparison between IL-1 beta and its antagonist IL-1ra, single or multiple residues along the IL-1 beta sequence were replace d 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-1 beta in three diffe rent biologic assays and in receptor binding, indicating that the intr oduced changes did not influence the functional structure of the prote in. 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 han d, two mutants showed diminished binding capacity with concomitant dec rease in biologic activity. Both mutants (SMIL-1, five substitutions i n 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 alle gedly 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 an d in vivo, but it retained full capacity of binding to IL-1R(II) (CDw1 21b) and acted as a selective antagonist of IL-1R(II). From these resu lts the following conclusions can be drawn. IL-1 beta binds to IL-1R(I ) and to IL-1R(II) through different sites, and the loop 164-173 appea rs as one of the areas involved in the selective interaction with IL-1 R(I). Agonist (IL-1 beta) and nonagonist (IL-1ra) binding to IL-1R(I) occur through distinct sites, with loops 164-173 and 202-214 of IL-1 b eta identified as two of the sites selectively involved in agonist bin ding to the activating receptor.