Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding.
In order to identify residues required for the binding of interleukin-8 (IL-8) to its receptor, mutants were constructed in which clusters of charged amino acids were systematically replaced with alanine along the entire IL-8 sequence. The mutants were tested for their ability to induce a receptor-mediated rise in cytosolic free Ca2+, a property of wild-type IL-8 which can readily be detected by flow cytometry using neutrophils loaded with the calcium probe Indo-1. Eleven of the 12 mutants caused neutrophil calcium mobilization at 5 nM; the exception being a triple alanine mutant at positions K3, E4, and R6, which was inactive at all concentrations tested (150 nM maximum). A second set of mutants was generated in which residues 1-15 were individually mutated to alanine. Mutants E4A, L5A, or R6A were all inactive in the Ca2+ assay at 5 nM and competed poorly with 125I-IL-8 for neutrophil receptor binding; I10A, E4A, L5A, and R6A had approximately 30-, 100-, 100-, and 1000-fold reduced affinity, as compared with control IL-8, respectively. The nuclear magnetic resonance structure of IL-8 indicates that, in solution, the side chains of E4, L5, R6, and I10 point away from the core of the protein and do not participate in any intramolecular hydrogen bonds or salt bridges (Clore, G. M., and Gronenborn, A. M. (1991) J. Mol. Biol. 217, 611-620).
Department of Cardiovascular Research, Genentech Inc., South San Francisco, California 94080.
C A
CA
Hébert
R V
RV
Vitangcol
J B
JB
Baker
D000595:Amino Acid Sequence; D000818:Animals; D003198:Computer Simulation; D016209:Interleukin-8 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000378:metabolism; D023062:Receptors, Interleukin-8A; D014961:X-Ray Diffraction
D016209:Interleukin-8; D011971:Receptors, Immunologic; D023062:Receptors, Interleukin-8A