Soluble mimics of a chemokine receptor: Chemokine binding by receptor elements juxtaposed on a soluble scaffold

Despite the broad biological importance of G protein–coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have...

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Bibliographic Details
Published in:Protein science 2003-11, Vol.12 (11), p.2482-2491
Main Authors: Datta, Amita, Stone, Martin J.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Binding Sites
Cell Line
Chemokine
Chemokine CCL11
chemokine receptor
Chemokines - chemistry
Chemokines - metabolism
Chemokines, CC - chemistry
Disulfides - chemistry
G protein‐coupled receptor
Ligands
Molecular Mimicry
Molecular Sequence Data
Protein Binding
protein chimera
protein design
protein‐protein interactions
Receptors, CCR3
Receptors, Chemokine - chemistry
Receptors, Chemokine - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
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Summary:Despite the broad biological importance of G protein–coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have developed a soluble protein that mimics ligand recognition by a GPCR. This receptor analog, dubbed CROSS5, consists of the N‐terminal and third extracellular loop regions of CC chemokine receptor 3 (CCR3) displayed on the surface of a small soluble protein, the B1 domain of Streptococcal protein G. CROSS5 binds to the CCR3 ligand eotaxin with a dissociation equilibrium constant of 2.9 ± 0.8 μM and competes with CCR3 for eotaxin binding. Control proteins indicate that juxtaposition of both CCR3 elements is required for optimal binding to eotaxin. Moreover, the affinities of CROSS5 for a series of eotaxin mutants are highly correlated with the apparent affinities of CCR3 for the same mutants, demonstrating that CROSS5 uses many of the same interactions as does the native receptor. The strategy used to develop CROSS5 could be applied to many other GPCRs, with a variety of potential applications.
ISSN:0961-8368
1469-896X
DOI:10.1110/ps.03254303