Probing receptor binding activity of interleukin-8 dimer using a disulfide trap.
Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.
Rajarathnam
Krishna
K
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555, USA. krrajara@utmb.edu
Prado
Gregory N
GN
Fernando
Harshica
H
Clark-Lewis
Ian
I
Navarro
Javier
J
D019943:Amino Acid Substitution; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D016209:Interleukin-8 / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D023062:Receptors, Interleukin-8A / Q000378:metabolism*; D023063:Receptors, Interleukin-8B / Q000378:metabolism*
D004220:Disulfides; D016209:Interleukin-8; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B