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

Abstract

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 GROγ, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the GROγ sequences corresponding to either the amine-terminal loop (amine acids 1-18) or the first β-sheet (amine acids 18-32) reduced binding to both IL8R1 and IL8R2. The third β-sheet of IL-8 (amine acids 46- 53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second β-sheet (amine acids 32-46) or the carboxylterminal α-helix (amine acids 53-72) had no significant effect. When IL-8 sequences were substituted into GROγ, a single domain containing the second β-sheet of IL-8 (amine acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. The amine-terminal loop (amine acids 1-18) and the third β-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 amine acid substitutions were made at positions where IL-8 and GROγ 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 Tyr13, Ser14, Phe21, and Lys49 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 GROγ, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GROγ. 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 Glu4-Leu5-Arg6 region, can modulate receptor binding affinity and IL8R1 specificity.