A structural and dynamic model for the interaction of interleukin-8 and glycosaminoglycans: support from isothermal fluorescence titrations.

Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8 > IL-8(H18A) >> IL-8(R68A) > IL-8(K67A) >> IL-8(K20A) > IL-8(R60A) >> IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.

Different affinities of glycosaminoglycan oligosaccharides for monomeric and dimeric interleukin-8: a model for chemokine regulation at inflammatory sites.

The binding of interleukin-8 (IL-8) to heparan sulfate (HS) proteoglycans on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Fluorescence anisotropy measurements yielded an IL-8 dimerization constant of 120 nM. The binding affinities, obtained by isothermal fluorescence titration, of size-defined heparin and HS oligosaccharides to the chemokine were found to depend on the oligomerization state of IL-8: high affinity was detected for monomeric and low affinity was detected for dimeric IL-8, referring to a self-regulatory mechanism for its chemoattractant effect. The highest affinity for monomeric IL-8 was detected for the HS octamer with a K(d) < 5 nM whereas the dissociation constants of dimeric IL-8 were found in the medium micromolar range. No indication for increasing affinities for monomeric IL-8 with increasing oligosaccharide chain length was found. Instead, a periodic pattern was obtained for the dissociation constants of the GAG oligosaccharides with respect to chain length, referring to optimum and least optimum chain lengths for IL-8 binding. GAG disaccharides were identified to be the minimum length for chemokine binding. Conformational changes of the dimeric chemokine, determined using CD spectroscopy, were detected only for the IL-8/HS complexes and not for heparin, pointing to an HS-induced activation of the chemokine with respect to receptor binding. Thermal unfolding of IL-8 yielded a single transition at 56 degrees C which was completely prevented by the presence of undigested HS or heparin, indicating structural stabilization, thereby prolonging the biological effect of the chemokine.