Monomeric variants of IL-8: effects of side chain substitutions and solution conditions upon dimer formation.
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 microM to mM, as compared with a dimerization constant of about 10 microM 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 Ca(2+)-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 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 microM, with consequences in vivo that are yet to be determined.
Lowman
H B
HB
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA. hbl@gene.com
Fairbrother
W J
WJ
Slagle
P H
PH
Kabakoff
R
R
Liu
J
J
Shire
S
S
Hébert
C A
CA
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D001704:Biopolymers; D006801:Humans; D016209:Interleukin-8 / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D018123:Receptors, Interleukin / Q000737:chemistry / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D012996:Solutions; D014961:X-Ray Diffraction
D015703:Antigens, CD; D001704:Biopolymers; D016209:Interleukin-8; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D012996:Solutions