Probing receptor binding activity of interleukin-8 dimer using a disulfide trap

Krishna Rajarathnam, Gregory N. Prado, Harshica Fernando, Ian Clark-Lewis, Javier Navarro

Research output: Contribution to journalArticlepeer-review

56 Scopus citations


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 ∼1000-fold difference between monomer-dimer equilibrium constant (μM) 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 ∼70-fold and CXCR2 with ∼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 μM 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.

Original languageEnglish (US)
Pages (from-to)7882-7888
Number of pages7
Issue number25
StatePublished - Jun 27 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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