A structural and dynamic model for the interaction of interleukin‐8 and glycosaminoglycans: Support from isothermal fluorescence titrations

Abstract

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.

Molecular Requirements for Sorting of the Chemokine Interleukin-8/CXCL8 to Endothelial Weibel-Palade Bodies

“…Mutations were performed to replace the GAG-binding residues (R20A/R60A/K65A/K67A/ R68A), the cysteine residues (C7S/C9S/C34S/C50S), and the residues encompassing the 3 10 on the solvent-exposed surface (33,35,37), they do have notable differences. For example, both helices interact with GAGs, but the main GAG-binding sites are located in different positions (30,38,39 20 ) with alanine did not affect sorting, suggesting that the positively charged residues of the ␣-helix involved in GAG binding are not important for targeting to WPB. It cannot be excluded that the remaining surface-exposed residues of the ␣-helix contribute to sorting by interaction with specific receptor molecules.…”

IL-8 Dictates Glycosaminoglycan Binding and Stability of IL-18 in Cystic Fibrosis

“…However, IL-8 greatly differs from IL-18 at both the sequence and structural level. Amino acids involved in IL-8-GAG binding have been identified as the basic amino acids His-18, Arg-60, Lys-64, Lys-67, and Arg68, located in the Cterminal helix and Lys20 in the proximal loop (48). The rank order of these residues' contributions to GAG binding is Lys-64, Arg-60, Lys-20, Lys-67, Arg-68, and His18.…”

Application of drug discovery software to the identification of heparin-binding sites on protein surfaces: a computational survey of the 4-helix cytokines

“…2 Residues containing atoms within 3.5 Å of ligand in the 5 lowest energy complexes ... [1] ... [3] Page 17 of 34 http://mc.manuscriptcentral.com/tandf/jenmol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 2 Residues containing atoms within 3.5 Å of ligand in the 5 lowest energy complexes Table 3: Prediction of heparin binding to the interferon/IL10 group of 4-helical cytokines. ... [7] ... [19] ... [15] ... [20] ... [18] ... [17] ... [21] ... [5] ... [22] ... [6] ... [16] ... [13] ... [24] ... [8] ... [25] ... [23] ... [14] ... [26] ... [9] ... [27] ... …”

“…Several groups have developed protocols for a systematic search of the entire surface of a protein to find the best heparin binding site [20][21][22]. We have used a combination of predictive docking with experimental studies in the successful identification of several heparin binding sites [23][24][25].…”