Chemokine-Binding Specificity of Soluble Chemokine-Receptor Analogues:  Identification of Interacting Elements by Chimera Complementation

• Published in: Biochemistry (Easton) 2004-11, Vol.43 (46), p.14602-14611
• Main Authors: Datta-Mannan, Amita, Stone, Martin J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Chemokine CCL11; Chemokine CCL2 - metabolism; Chemokines, CC - genetics; Chemokines, CC - metabolism; Models, Molecular; Molecular Mimicry; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Receptors, CCR2; Receptors, CCR3; Receptors, Chemokine - genetics; Receptors, Chemokine - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Solubility
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi048990e

The specificity of chemokine-receptor interactions plays a central role in the regulation of leukocyte migration in inflammatory responses. Herein, we describe a soluble mimic of CC chemokine receptor 2 (CCR2), dubbed CROSS−N2E32, which incorporates the N-terminal region (N) and third extracellular loop (E3) elements of CCR2 displayed on the surface of a soluble protein scaffold. CROSS−N2E32 binds to the CCR2 ligand monocyte chemoattractant protein-1 (MCP-1) with a dissociation equilibrium constant of 1.1 ± 0.1 μM but does not bind to the cognate chemokines of the receptor CCR3 (eotaxin-1, -2, and -3). Similarly, a soluble analogue of CCR3 (CROSS5−N3E33) binds to eotaxin-1, -2, and -3 but not to MCP-1. Thus, these receptor analogues have the same specificity as the natural receptors. Using soluble proteins containing N and E3 elements from different receptors (CROSS−N2E33 and CROSS−N3E32), we demonstrate that both receptor elements are required for optimal binding to the cognate chemokines. In addition, we report the binding affinities of all four CROSS proteins to a panel of two wild-type and six chimeric chemokines. These complementation studies indicate the regions of the chemokines that interact with each element of the receptors, allowing us to deduce the orientations of the receptor extracellular elements relative to the bound chemokines.