Mapping Receptor Binding Sites in Interleukin (IL)-1 Receptor Antagonist and IL-1β by Site-directed Mutagenesis

Abstract

Interleukin-l receptor antagonist (IL-lra), an ILl family member, binds with high affinity to the type I ILl receptor (IL-lRI), blocking ILl binding but not inducing an ILl like response. Extensive site-directed mutagenesis has been used to identify residues in IL-lra and IL-lf3 involved in binding to IL-IRI. These analyses have revealed the presence of two discrete receptor binding sites on IL-lf3. Only one of these sites is present on IL-lra, consisting of residues Trp-16, Gin-20, Tyr-34, GIn-36, and Tyr-147. Interestingly, the absent second site is at the location of the major structural difference between IL-lra and IL-lf3, which are otherwise structurally similar. The two receptor binding sites on IL-lf3 are also present on IL-la. Thus, it appears that the two ILl agonist molecules have two sites for IL-IRI binding, and the homologous antagonist molecule, IL-lra, has only one. Based on these observations, a hypothesis is presented to account for the difference in activity between the agonist and antagonist proteins. It is proposed that the presence of the two receptor binding sites may be necessary for agonist activity. The IL-11 family of proteins are related by sequence similarity , gene organization, and three-dimensional structure (Eisenberg et at., 1990, 1991; Carter et at., 1990). The three proteins, IL-1a, IL-1{3, and IL-1ra, all exhibit a {3-trefoil topol-ogy characterized by six {3-strands forming a tapered {3-barrel, which is closed at the wide end by another six {3-strands (Murzin et at., 1992; Vigers et at., 1994). The two agonist proteins, IL-1a and IL-l{3, have similar biological activities, mediated through their high affinity interaction with the type 1 ILl receptor (lL-lRD (Sims et at., 1993). These two proteins are believed to play an important role in causing both local and systemic inflammatory responses (Dinarello, 1991). The third family member, IL-1ra, also binds with high affin