Diana Boraschi, Paola Bossù, Giovanni Macchia, Paolo Ruggiero & Aldo Tagliabue

Dept. Biotechnology, Research Center Dompé SpA, Via Campo di Pile, I-67100 L'Aquila Italy

Received 8/5/96; Accepted 9/11/96; On-line 11/01/96

3. DISCUSSION

Several studies have allowed in the last decade to clarify the structural characteristics of IL-1ß, as well as of IL-1alpha and, more recently, of IL-1ra and to put them in relation with their capacity to bind to the specific receptors and to exert biological effects. Some of these investigations have taken advantage of specifically mapped monoclonal antibodies and of synthetic peptides in order to define, on the protein surface, sites selectively important for different activities or for receptor binding capacity (reviewed in 9, 55). As a general finding, from these studies the hypothesis can be drawn that different sites of the protein could be involved in distinct functions and that binding sites can be separated from active sites.

3.1 Mutagenesis

The major body of information on the structure-function relationship in proteins of the IL-1 family comes, however, from mutagenesis studies, in which the biological relevance of restricted areas of the protein or even of single amino acids has been evaluated by residue substitution along the IL-1 sequence with techniques of genetic engineering (see also 9, 55). An extensive listing of single substitutions and multiple modifications, including deletions and insertions, is provided for human IL-1alpha (Table I and Table II), for human IL-1ß (Table III and Table IV) and for human IL-1ra (Table V). As it can be noticed, the large majority of data that are available refer to IL-1ß, rather than to IL-1alpha, because of the prominent role of this cytokine in the inflammatory events. Several observations can be made on these data. One important finding is that binding capacity for IL-1R_I does not correlate with binding to IL-1RII, suggesting that despite the structural similarity between the two receptors the establishment of contact with receptors involves different domains of the IL-1 protein. Another general impression from observation of these data is that, although binding capacity for IL-1R_I is often associated to expression of biological activity, this is not always true. In fact, mutations at certain sites (e.g. R127 G; 76, 82-87) do not significantly influence the IL-1RI-binding capacity, but can deeply affect the agonist capacity, at least for some of the observed biological effects. In view of the recently discovered role of IL-1RAcP in the establishment of optimal agonist binding of IL-1 to IL-1RI, this and other observations which dissociate binding to IL-1R_I from agonist effects should now be reconsidered. In fact, sites such as R127 can be selectively involved in the association of IL-1/IL-1RI complex to IL-1RAcP and therefore important for biological activity eventhough irrelevant to IL-1R_I binding. Along the same line, it should also be considered that the importance of receptor binding for distinct biological effects appears to vary considerably. Thus, some activities apparently require involvement of IL-1RAcP whereas others may only need IL-1R_I binding. Ultimately, certain data of parallel mutagenesis studies of IL-1ß and IL-1ra can help clarify the structural requirements for agonist vs. antagonist activity. For instance, the residue D261 of IL-1ß, corresponding to the residue K145 of IL-1ra, is apparently pivotal for agonist capacity and possibly for interaction with IL-1RAcP. In fact, replacement of D261 with K in IL-1ß does not affect binding but deeply impairs biological effects, whereas substitution of K145 with D in IL-1ra leaves binding unaltered but induces IL-1-like agonist capacity (56, 65, 82, 83, 98, 113). Another domain of high importance for agonist capacity of IL-1ß is the ß-bulge located between ß strands 4 and 5 (loop D) which, however, is not involved in receptor binding and therefore can possibly take part in interaction with IL-1RAcP. In fact, insertion of this domain within the IL-1ra protein leads to acquisition of partial agonist activity without significant variation of receptor binding (113). This finding is indeed in line with previous reports showing selective IL-1-like biological activity by synthetic fragments corresponding to loop D in the absence of binding to IL-1R_I (55, 114-118). It is thus hypothesized that the loop D of IL-1ß is responsible for interaction with IL-1RAcP and for the expression of immunostimulatory activity, whereas other areas of the molecule are more relevant to binding to IL-1R_I and to agonist inflammatory effects.

3.2 Structural analysis

Valuable information has been obtained from structural studies of IL-1 proteins, either from its crystallographic analysis or from NMR spectroscopy. Also in this case, analysis of IL-1ß structure is far more detailed and complete than the information available for other proteins of the family. Crystallization of IL-1ß and resolution of its 3D structure revealed a ß-barrel structure with pseudo 3-fold symmetry, formed by twelve ß-strands (14, 15, 119, 120). A large body of detailed structural information on IL-1ß in solution has been provided by a series of excellent NMR studies, and by some studies with circular dichroism, fluorescence and calorimetry, which have allowed complete assignment of backbone and side chains, identification and position of associated water molecules and kinetics of folding and unfolding of the IL-1ß protein (89, 121-130). Besides these studies on IL-1ß, other important investigations regard structural analysis of IL-1ß mutant proteins (81, 89, 94, 131), analysis of structural characteristics of IL-1alpha (16, 64) and of IL-1ra (18, 19, 132, 133). From all these data, a series of studies of computer-aided molecular modeling have been completed, which provide exhaustive information on the structural characteristics of agonist vs. antagonist IL-1 proteins in relation to their binding capacity and bioactivity (17, 72, 75, 83, 84, 96, 134, 135).

3.3 Models of ligand/receptor interaction

A summary of the models drawn from structural, conformational and biological data on IL-1 proteins could thus be provided. These models must, however, be proven true by experimental data, which are beginning to be available, on the crystallographic analysis of ligand/receptor complexes (136, 137). The available models also do not yet take into account the possible involvement of IL-1RAcP, which does not associate directly with IL-1 but which interacts with the IL-1/IL-1RI complex. One common site of binding to IL-1R_I can be defined on agonist IL-1ß and IL-1alpha and on antagonist IL-1ra. This area, defined as site A (83), located on the side of the ß-barrel structure of IL-1 proteins, includes a discontinuous cluster of residues which include H146, Q131, Q148 and R127 in IL-1ß (see also area II described in 75; and the hydrophilic binding area described in 96), N141 in IL-1alpha, and Y34, Q20, Q36, W16 and Y147 in IL-1ra (59, 75, 83, 85, 88, 96). Sites A of the three IL-1 proteins show remarkable structural similarity in molecular models and are considered to be responsible for the non-agonist binding with IL-1RI. This site, in fact, represents the only contact site of IL-1ra for IL-1RI, suggesting that interaction of IL-1 proteins with IL-1R_I through this area is not sufficient for cell triggering. In the case of agonist proteins, IL-1alpha and IL-1ß, another contact area for IL-1R_I has been identified which is missing in IL-1ra. This has been defined as site B and includes residues important for the biological activity of IL-1, i.e. R120, L122, F162, I172, K208, K209, K219, E221 in IL-1ß (see also discontinuous binding site described in 72; and area I described in 75); R128, I130, D176, D177, K212, N220, W225, G229 and Q248 in IL-1alpha (58, 72, 75, 83). Comparison of structural models of IL-1 proteins shows that site B, located at the open end of the ß-barrel, is present in the two agonist proteins, IL-1alpha and IL-1ß, but absent in the IL-1ra structure, which in this particular area shows the highest degree of dissimilarity with other IL-1 molecules (15-18, 83, 113). Thus, site B is considered responsible for agonist binding to IL-1RI. Initial crystallographic studies of the patterns of interaction between IL-1ra and IL-1R_I have shown that, in fact, the antagonist protein contacts the first and second Ig-like domain of IL-1RI, whereas no strong interaction is established with the third domain (137). Modeling of IL-1ß/IL-1RI complex on the ground of the IL-1ra/IL-1RI crystal analysis apparently indicates that the agonist IL-1 can interact, through a cluster of positively charged residues, with a negatively charged area in the third domain of IL-1R_I (137). In line with the identification of IL-1RAcP as a possibly important factor in determining agonist vs. non-agonist binding to IL-1R_I (IL-1RAcP can associate to IL-1alpha/IL-1RI and IL-1ß/IL-1RI complexes but not to the IL-1ra/IL-1RI complex; 36, 113). Thus, it can be proposed that, in the agonist action of IL-1, site A is responsible for the first contact with IL-1RI, whereas site B takes part in a second type of interaction which involves the recruitment of IL-1RAcP into the complex. In IL-1ra, where only site A is present, no involvement of IL-1RAcP is possible and therefore no cell activation can be initiated.

3.4 Perspective

The large body of information available from mutagenesis and structural studies, together with those from studies with specific antibodies and with synthetic or recombinant peptides, has allowed to define the first general models of interaction of proteins of the IL-1 family with their receptors. The definition of agonist interaction of IL-1alpha and IL-1ß with the activating receptor IL-1R_I will receive an enormous improvement from the recent identification of IL-1RAcP, the receptor accessory protein necessary for optimal interaction and receptor activation by agonist ligands. Further information will come from structural crystallographic analysis of IL-1/IL-1R complexes, to implement and clarify the present models.

Thus, it should be possible, within a short time, to unravel the precise features of agonist vs. non-agonist interactions of IL-1 proteins with their receptors, and to provide a solid background for the rational design of optimal antagonists for the therapeutic use. The study of interaction with the inhibitory receptor IL-1RII, which is still scanty in comparison to data gathered for IL-1RI, will also, in future, provide valuable information on the ways to control the IL-1 activities.