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

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 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.

SUBMITTER: Rajarathnam K

PROVIDER: S-EPMC2518044 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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

Rajarathnam Krishna K Prado Gregory N GN Fernando Harshica H Clark-Lewis Ian I Navarro Javier J

Biochemistry 20060601 25

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 ...[more]

PMID: 16784240

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Project description:The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here, we evaluate the binding and biological activity of a new class of pyrrolidinone and piperidinone compounds, along with derivatives of previously-identified pyrazole and propylamine compounds. Competition assays revealed that the compounds displace a fluorescently labeled peptide (AE147-FAM) with inhibition constant (Ki ) values ranging from 6 to 63??M. Structure-based computational pharmacophore analysis followed by extensive explicit-solvent molecular dynamics (MD) simulations and free energy calculations suggested the pyrazole-based and piperidinone-based compounds adopt different binding modes, despite their similar two-dimensional structures. In cells, pyrazole-based compounds showed significant inhibition of breast adenocarcinoma (MDA-MB-231) and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but piperidinone-containing compounds exhibited no cytotoxicity even at concentrations of 100??M. One pyrazole-based compound impaired MDA-MB-231 invasion, adhesion, and migration in a concentration-dependent manner, while the piperidinone inhibited only invasion. The pyrazole derivative inhibited matrix metalloprotease-9 (gelatinase) activity in a concentration-dependent manner, while the piperidinone showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1? and NF-?B signaling, while pyrrolidinones and piperidinones had no effect. Annexin?V staining suggested that the effect of the pyrazole-based compound on proliferation was due to cell killing through an apoptotic mechanism. The compounds identified represent valuable leads in the design of further derivatives with higher affinities and potential probes to unravel the protein-protein interactions of uPAR.

Dataset Information

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

Publications

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

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