Characterization of the interactions between interleukin-8 and soluble mimics of the chemokine receptors CXCR1 and CXCR2

Chemokines are a group of proteins that play important roles in initiating and mediating various inflammatory and immune responses. The 'CXC' chemokine interleukin-8 (IL-8) mediates these responses through interactions with its cognate receptors, CXCR1 and CXCR2. Chemokine receptors are seven transmembrane helix-containing, G protein-coupled receptors. In order to obtain structural insights, while bypassing the difficulties working with the intact receptors, we have previously developed a soluble protein system that mimics the ligand-binding elements of chemokine receptors (1). These soluble mimics, designated by the acronym CROSS (chemokine receptor elements on a soluble scaffold), have been used previously to study the interactions of 'CC' chemokine receptors with their cognate chemokines (1, 2). This work applies the same approach to the 'CXC' family of chemokine receptors. I describe the design, expression, and characterization, of CROSS-NX1E3X1 and CROSS-N X2E3X2, which are soluble proteins displaying the N-terminal and E3 loop elements of CXCR1 and CXCR2 respectively. Isothermal titration calorimetry has been used to determine the binding affinities of IL-8 with CROSS-NX1E3X1 and CROSS-NX2E3 X2 as ∼800 nM and ∼6 muM respectively. Additionally, NMR was used to determine the structural contribution of each receptor element, by mapping the binding sites of these CROSS proteins on IL-8.;1Datta, A., and Stone, M.J. (2003) Protein Science. 12: 2482-91. 2Datta-Mannan, A., and Stone, M.J. (2004) Biochemistry. 43: 14602-11.