Structural requirements for interleukin-8 function identified by design of analogs and CXC chemokine hybrids.

Structure-activity relationships of human interleukin-8 (IL-8) were probed using chemically synthesized analogs with single or double amino acid substitutions, as well as hybrids derived by substituting IL-8 regions into IP10, a related protein that lacks IL-8 activity. The analogs were tested for functional activity by measuring induction of elastase release from human neutrophils and competition for binding of radiolabeled IL-8. The hybrid studies indicated that Gly31 and Pro32, as well as the NH2-terminal region from IL-8 are required to convert IP10 into a fully functional protein, suggesting that these elements are critical for IL-8 activity. Both disulfide bridges, linking residue 7 to 34 and residue 9 to 50, were critical for function, as shown by substituting the cysteine pairs with alpha-aminobutyric acid. Single conservative substitutions were generally accepted into the 10-22 region of IL-8, which contrasts with the ELR motif (residues 4-6), previously shown to be essential for activity. The importance of residues within the 10-15 region and the 17-22 region was demonstrated with hybrids. In addition, some of the 4-22 residues have structural roles that may be important; for example, Tyr13, Phe17, and Phe21 are involved in aromatic interactions in the IL-8 structure, and are also moderately sensitive to modification. Except for Cys50, the results argue against a role for the 36-72 region, including the COOH-terminal alpha-helix, in receptor binding. We conclude that the disulfide bridges and 30-35 turn provide a structural scaffold for the NH2-terminal region which includes the primary receptor-binding site (the ELR motif) and secondary binding and conformational determinants between residues 10 and 22.