Mutagenesis studies of interleukin-8. Identification of a second epitope involved in receptor binding.
Interleukin-8 (IL-8) is a dimeric, C-X-C chemokine, produced by a variety of cells and which elicits proinflammatory responses from the neutrophil. As a prelude to drug design, we have investigated the interactions between IL-8 and its receptor by preparing a number of single-site mutants of IL-8 and determining their activity in receptor-binding and functional assays. In order to define the binding surface as precisely as possible, we have used chemical shifts obtained from nuclear magnetic resonance spectroscopy to screen mutant proteins for structural changes which affect regions of the IL-8 surface remote from the site of mutation. In addition to a previously recognized sequence, Glu4-Leu5-Arg6 in the N-terminal peptide, we have identified a second epitope comprising a contiguous group of non-sequential, solvent-exposed, hydrophobic residues, Phe17, Phe2l, Ile22, and Leu43. These two receptor-binding regions are separated by over 20 A in the IL-8 structure and are important both for receptor binding and function. In addition, we have shown through the production of a covalently linked IL-8 dimer, that subunit dissociation is not necessary for biological activity.
Williams
G
G
Roche Research Centre, Welwyn Garden City, Hertfordshire, United Kingdom.
Borkakoti
N
N
Bottomley
G A
GA
Cowan
I
I
Fallowfield
A G
AG
Jones
P S
PS
Kirtland
S J
SJ
Price
G J
GJ
Price
L
L
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000097:blood; D002462:Cell Membrane / Q000276:immunology; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine; D007930:Leucine; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000276:immunology / Q000502:physiology*; D010649:Phenylalanine; D017433:Protein Structure, Secondary; D018123:Receptors, Interleukin / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D000939:Epitopes; D016209:Interleukin-8; D046911:Macromolecular Substances; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins; D007532:Isoleucine; D010649:Phenylalanine; D007930:Leucine; D002118:Calcium