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 journalArticle

44 Citations (Scopus)

Abstract

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
JournalBiochemistry
Volume45
Issue number25
DOIs
StatePublished - Jun 27 2006

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Interleukin-8
Disulfides
Dimers
Monomers
Interleukin-8A Receptors
Interleukin-8B Receptors
Ligands
Dimerization
G-Protein-Coupled Receptors
Chemokines
Neutrophils
Equilibrium constants
Nuclear magnetic resonance
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Probing receptor binding activity of interleukin-8 dimer using a disulfide trap. / Rajarathnam, Krishna; Prado, Gregory N.; Fernando, Harshica; Clark-Lewis, Ian; Navarro, Javier.

In: Biochemistry, Vol. 45, No. 25, 27.06.2006, p. 7882-7888.

Research output: Contribution to journalArticle

Rajarathnam, Krishna ; Prado, Gregory N. ; Fernando, Harshica ; Clark-Lewis, Ian ; Navarro, Javier. / Probing receptor binding activity of interleukin-8 dimer using a disulfide trap. In: Biochemistry. 2006 ; Vol. 45, No. 25. pp. 7882-7888.
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