A molecular switch of chemokine receptor selectivity. Chemical modification of the interleukin-8 Leu25 --> Cys mutant.
Interleukin-8 (IL-8), a member of the CXC chemokine family, is a key activator of neutrophils. We have previously shown that two novel CC chemokine-like properties, namely monocyte chemoattraction and binding to CC CKR-1, are introduced into IL-8 by mutating Leu25 to the conserved tyrosine present in CC chemokines. To further investigate the role of this position in receptor selectivity, we have mutated Leu25 to cysteine. The protein folds correctly with two disulfide bonds and a free thiol group at Cys25. This mutant behaves overall like wild-type IL-8, with little change in neutrophil chemotaxis and IL-8 receptor binding, and has no effect on CC CKR-1. These data are consistent with cysteine being approximately isosteric with the natural amino acid leucine. However, modification of the cysteine by addition of a fluorescent N-methyl-N-(2-N-methyl, N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)aminoethyl)acetamido (NBD) group lowers potency in neutrophil chemotaxis and affinity in IL-8 receptor binding assays by 2 orders of magnitude. This Leu25 --> Cys-NBD mutant introduces monocyte chemoattractant activity and the ability to displace 125I-labeled macrophage inflammatory protein-1 alpha from the recombinant CC CKR-1 receptor. Additionally, we show a specific interaction between the fluorescent mutant and the N-terminal 34-amino acid peptide from CC CKR-1. This confirms the importance of this region in IL-8 in receptor binding and in conferring specificity between CXC and CC chemokines. Circular dichroism spectra of the IL-8 mutants having CC chemokine-like activity show a consistent drop in 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.
Lusti-Narasimhan
M
M
Glaxo Institute for Molecular Biology, 1228 Plan-les-Ouates, Geneva, Switzerland.
Chollet
A
A
Power
C A
CA
Allet
B
B
Proudfoot
A E
AE
Wells
T N
TN
D009327:4-Chloro-7-nitrobenzofurazan; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D018946:Chemokine CCL5 / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D018922:HL-60 Cells; D006801:Humans; D016209:Interleukin-8 / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007930:Leucine; D008969:Molecular Sequence Data; D009000:Monocytes / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D018123:Receptors, Interleukin / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection
D015703:Antigens, CD; D018946:Chemokine CCL5; D017931:DNA Primers; D016209:Interleukin-8; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011994:Recombinant Proteins; D009327:4-Chloro-7-nitrobenzofurazan; D007930:Leucine; D003545:Cysteine