A structural and dynamic model for the interaction of interleukin-8 and glycosaminoglycans: Support from isothermal fluorescence titrations
A structural and dynamic model for the interaction of interleukin-8 and glycosaminoglycans: Support from isothermal fluorescence titrations
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DOI：
10.1002/prot.10590
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发表时间：
2004-03-01
影响因子：
2.9
通讯作者：
Kungl, AJ
中科院分区：
文献类型：
--
作者：
Krieger, E;Geretti, E;Kungl, AJ
白细胞介素-8（IL-8）与内皮细胞表面糖胺聚糖（GAG）的结合对于中性粒细胞向炎症部位的募集至关重要。由于GAG的高度灵活性和尺寸，从计算机对接实验中推导出关于这种相互作用的结构知识已被证明是困难的。因此，我们开发了一种对接方法，考虑到配体和蛋白质的灵活性，通过运行类似于15,000个分子动力学模拟的对接事件与不同的初始方向的结合伙伴。该方法显示出成功地再现了参与GAG结合的碱性成纤维细胞生长因子的残基。肝素六糖与IL-8的对接得到涉及碱性残基His 18、Lys 20、Arg 60、Lys 64、Lys 67和Arg 68的相互作用界面。通过对其中这些氨基酸被丙氨酸取代的IL-8单位点突变体进行等温荧光滴定，确定对肝素的亲和力为：IL-8 > IL-8（H18 A）远大于IL-8（R68 A）> IL-8（K67 A）远大于IL-8（K20 A）> IL-8（R60 A）远大于IL-8（K64 A）。与从模型计算的结合能的比较揭示了wtIL 8和H18 A突变体的高值，以及其余突变体的显著较低但相似的能量。通过N-乙酰化十二糖连接与二聚体趋化因子中的两个IL-8单体中的每一个结合的两个完全硫酸化的六糖，得到复杂的结构，其中GAG分子以平行方式与IL-8的N-末端α-螺旋对齐，如马蹄铁。一个5-ns的分子动力学模拟这个复合物证实了它的结构稳定性，并揭示了在两个结合位点的重新定位，其中一个二糖成为中央结合单位。使用不同硫酸化肝素二糖的等温荧光滴定实验证实，单个二糖确实可以以高亲和力结合IL-8。(C)2004 Wiley-Liss，Inc.
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 similar to15,000 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) much greater than IL-S(R68A) > IL-8(K67A) much greater than IL-8(K20A) > IL-8(R60A) much greater than 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 o 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. (C) 2004 Wiley-Liss, Inc.