Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket

To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity, Receptor binding activity was tested by competition of I-125-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines, Substitution into IL-8 of the GRO gamma sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2, The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2, Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect, When IL-8 sequences were substituted into GRO gamma, a single domain containing the second beta-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2, The amino-terminal loop (amino acids 1-18) and the third beta-sheet (amino acids 46-53) of IL-8 had little effect when substituted individually but showed increased binding to both receptors when substituted in combination. Individual amino acid substitutions were made at positions where IL-8 and GRO gamma sequences differ within the regions of residues 11-21 and 46-53, IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1, Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2, A combined mutation Y13L/S14Q selectively decreased binding to IL8R2, Residues Tyr(13), Ser(14), Phe(21), and Lys(49) are clustered in and around a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence, A homology model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GRO gamma, These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic pocket itself may be essential for IL8R1 binding, Thus this region contains a second site for IL-8 receptor recognition that, in combination with the Glu(4)-Leu(5)-Arg(6) region, can modulate receptor binding affinity and IL8R1 specificity.