A MOLECULAR SWITCH OF CHEMOKINE RECEPTOR SELECTIVITY - CHEMICAL MODIFICATION OF THE INTERLEUKIN-8 LEU(25)-]CYS MUTANT

Interleukin-8 (IL-8), a member of the CXC chemokine family, is a key a ctivator of neutrophils. We have previously shown that two novel CC ch emokine-like properties, namely monocyte chemoattraction and binding t o CC CKR-1, are introduced into IL-8 by mutating Leu(25) to the conser ved tyrosine present in CC chemokines. To further investigate the role of this position in receptor selectivity, we have mutated Leu(25) to cysteine. The protein folds correctly with two disulfide bonds and a f ree thiol group at Cys(25). This mutant behaves overall like wild-type IL-8, with little change in neutrophil chemotaxis and IL-8 receptor b inding, and has no effect on CC CKR-1. These data are consistent with cysteine being approximately isosteric with the natural amino acid leu cine, However, modification of the cysteine by addition of a fluoresce nt N-methyl-N-(2-N-methyl, trobenz-2-oxa-1,3-diazol-4-yl)aminoethyl)ac etamido (NBD) group lowers potency in neutrophil chemotaxis and affini ty in IL-8 receptor binding assays by 2 orders of magnitude. This Leu( 25) --> Cys-NBD mutant introduces monocyte chemoattractant activity an d the ability to displace I-125-labeled macrophage inflammatory protei n-1 alpha from the recombinant CC CKR-1 receptor. Additionally, we sho w a specific interaction between the fluorescent mutant and the N-term inal 34-amino acid peptide from CC CKR-1. This confirms the importance of this region in IL-8 in receptor binding and in conferring specific ity between CXC and CC chemokines. Circular dichroism spectra of the I L-8 mutants having CC chemokine-like activity show a consistent drop i n alpha-helical content compared with the spectra for wild-type IL-8. This suggests that distortion of the C-terminal helix may play a role in chemokine receptor-ligand selectivity.