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Monomeric variants of IL‐8: Effects of side chain substitutions and solution conditions upon dimer formation
Author(s) -
Lowman Henry B.,
Fairbrother Wayne J.,
Slagle Paul H.,
Kabakoff Rhona,
Hebert Caroline A.,
Liu Jun,
Shire Steven
Publication year - 1997
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560060309
Subject(s) - dimer , chemistry , monomer , wild type , stereochemistry , receptor , affinities , biophysics , biochemistry , mutant , biology , organic chemistry , gene , polymer
IL‐8 dimers have been observed in NMR and X‐ray structures of the protein. We have engineered IL‐8 monomers by mutations of residues throughout the dimer interface, which introduce hindrance determinants to dimerization. These IL‐8 variants are shown by NMR to have wild‐type monomer folding, but by ultracentrifugation to have a range of dimerization constants from /xM to mM, as compared with a dimerization constant of about 10 /xM for wild‐type IL‐8, under physiological salt and temperature conditions. The monomeric variants of IL‐8 bind the erythrocyte chemokine receptor DARC, as well as the neutrophil IL‐8 receptors CXCR1 and CXCR2 with affinities similar to that of wild‐type IL‐8. In addition, the monomeric variants were shown to have agonist activity, with similar potency to wild‐type, in both Ca2+‐flux assays on CXCR1 and CXCR2 transfected cells, and in chemotaxis assays on neutrophils. Thus, these variants confirm that monomeric IL‐8 is functionally equivalent to wild‐type in in vitro assays. We have also investigated the effects of various solution conditions upon IL‐8 dimer formation using analytical ultracentrifugation. At salt concentrations, temperatures, and pH conditions lower than physiological, the dimerization affinity of IL‐8 is greatly enhanced. This suggests that, under some conditions, IL‐8 dimer formation may occur at concentrations of IL‐8 considerably lower than 10 fiM, with consequences in vivo that are yet to be determined.