Biophysical investigation of a ternary chemokine signalling complex

V. Wabitsch, H. Wutschek, K. Mechtler*, and A. J. Kungl

Institute of Pharmaceutical Sciences, University of Graz, A-8010 Graz, Austria; *I.M.P., Research Institute of Molecular Pathology, A-1030 Vienna, Austria

The chemokines comprise a family of 8-10 kDa proteins which can be subdivided into constitutive and inflammatory or inducible signalling proteins. The pro-inflammatory chemokine Interleukin-8 (IL-8) belongs to the CXC ELR subfamily and signals by binding to CXCR1 and CXCR2 which are seven-transmembrane G-protein-coupled receptors preferentially expressed on neutrophils. Activation of the receptor(s) results in conversion of the selectin-mediated rolling of the neutrophil into firm integrin-mediated adhesion, change of shape and extravasation to the site of inflammation. This process is mediated by glycosaminoglycans (GAGs) which are polyanionic (sulfated) linear polysaccharides consisting of the recurring disaccharide units hexuronic acid and D-glucosamine. GAGs are components of the cell surface and extracellular matrix proteoglycans. They mediate chemokine function by immobilising them on the endothelial cell surface leading to the establishment of a chemotactic gradient, thereby presenting the chemokine to the circulating neutrophils. Here we present first results on the biophysical investigation of the ternary complex consisting of IL-8, GAG, and the (synthetic) ectodomain of CXCR1. Fluorescence isothermal titration experiments and gel mobility assays were used to study the mutual dependence of affinities of the three complex components. Far-UV circular dichroism provided insight into secondary structural changes upon component binding. In addition, fluorescence anisotropy revealed quaternary structure formation/disruption induced by component binding. Since the ternary chemokine signalling complex represents the actual therapeutic target for IL-8-mediated inflammation (such as rheumatoid arthritis), the biophysical data presented here provide a quantitative measure for rational inhibitor development.

Email: andreas.kungl@uni-graz.at

Address: Institute of Pharmaceutical Sciences, Protein Chemistry and Biophysics Group, University of Graz, Universitaetsplatz 1, 8010 Graz, Austria