University of California, 5200 North Lake Road, Merced, California 95343, USA.
Biochemistry. 2010 Aug 24;49(33):7012-22. doi: 10.1021/bi100549y.
Chemokines are important immune proteins, carrying out their function by binding to glycosaminoglycans (GAGs) on the endothelial surface and to cell surface chemokine receptors. A unique viral chemokine analogue, viral macrophage inflammatory protein-II (vMIP-II), encoded by human herpesvirus-8, has garnered interest because of its ability to bind to multiple chemokine receptors, including both HIV coreceptors. In addition, vMIP-II binds to cell surface GAGs much more tightly than most human chemokines, which may be the key to its anti-inflammatory function in vivo. The goal of this work was to determine the mechanism of binding of GAG by vMIP-II. The interaction of vMIP-II with a heparin-derived disaccharide was characterized using NMR. Important binding sites were further analyzed by mutagenesis studies, in which corresponding vMIP-II mutants were tested for GAG binding ability using heparin chromatography and NMR. We found that despite having many more basic residues than some chemokines, vMIP-II shares a characteristic binding site similar to that of its human analogues, utilizing basic residues R18, R46, and R48. Interestingly, a particular mutation (Leu13Phe) caused vMIP-II to form a pH-dependent CC chemokine-type dimer as determined by analytical ultracentrifugation and NMR. To the best of our knowledge, this is the first example of engineering a naturally predominantly monomeric chemokine into a dissociable dimer by a single mutation. This dimeric vMIP-II mutant binds to heparin much more tightly than wild-type vMIP-II and provides a new model for studying the relationship between chemokine quaternary structure and various aspects of function. Structural differences between monomeric and dimeric vMIP-II upon GAG binding were characterized by NMR and molecular docking.
趋化因子是重要的免疫蛋白，通过与内皮表面的糖胺聚糖 (GAG) 和细胞表面趋化因子受体结合来发挥其功能。一种独特的病毒趋化因子类似物，人疱疹病毒 8 编码的病毒巨噬细胞炎性蛋白-II (vMIP-II)，因其能够结合多种趋化因子受体而引起关注，包括 HIV 核心受体。此外，vMIP-II 与细胞表面 GAG 的结合比大多数人类趋化因子紧密得多，这可能是其在体内抗炎功能的关键。这项工作的目的是确定 vMIP-II 与 GAG 结合的机制。使用 NMR 表征了 vMIP-II 与肝素衍生二糖的相互作用。通过突变研究进一步分析了重要的结合位点，其中相应的 vMIP-II 突变体通过肝素层析和 NMR 测试了 GAG 结合能力。我们发现，尽管 vMIP-II 比一些趋化因子具有更多的碱性残基，但它与人类类似物共享一个特征性结合位点，利用碱性残基 R18、R46 和 R48。有趣的是，一个特定的突变（Leu13Phe）导致 vMIP-II 在分析超速离心和 NMR 测定时形成 pH 依赖性 CC 趋化因子型二聚体。据我们所知，这是通过单个突变将天然主要为单体的趋化因子工程化为可分离二聚体的第一个例子。这种二聚体 vMIP-II 突变体与肝素的结合比野生型 vMIP-II 紧密得多，为研究趋化因子四级结构与功能各个方面之间的关系提供了新模型。通过 NMR 和分子对接表征了 GAG 结合时单体和二聚体 vMIP-II 之间的结构差异。
