HEPARIN-BINDING TO PLATELET FACTOR-IV - AN NMR AND SITE-DIRECTED MUTAGENESIS STUDY - ARGININE RESIDUES ARE CRUCIAL FOR BINDING
HEPARIN-BINDING TO PLATELET FACTOR-IV - AN NMR AND SITE-DIRECTED MUTAGENESIS STUDY - ARGININE RESIDUES ARE CRUCIAL FOR BINDING
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DOI：
10.1042/bj3120357
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发表时间：
1995-12-01
影响因子：
4.1
通讯作者：
DALY, TJ
中科院分区：
文献类型：
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作者：
MAYO, KH;ILYINA, E;DALY, TJ
天然血小板因子 4 (PF4) 是一种不对称相关的同源四聚体蛋白（70 个残基/亚基），已知可结合肝素等多硫酸化糖胺聚糖，而 PF4 N 末端嵌合突变体 M2 (PF4-M2) 则形成对称四聚体 [Mayo、Roongta、Ilyina、Milius、Barker、Quinlan、La Rosa 和 Daly (1995) 生物化学34, 11399-11409] 使用这种 32 kDa 蛋白质进行 NMR 研究。此外，PF4-M2 与肝素的结合亲和力与天然 PF4 相似。这里提供的 NMR 数据表明，肝素（9000 Da 截止值）与 PF4-M2 结合，虽然不会扰乱蛋白质的整体结构，但会扰乱特定的侧链质子共振，该共振映射到四聚体 PF4-M2 表面带正电侧链环内的空间相关残基。与以 C 端 a 螺旋赖氨酸为中心的 PF4-肝素结合模型相反，这项研究表明，包含 Arg-20、Arg-22、His-23 和 Thr-25 以及 Lys-46 和 Arg-49 的环更容易受到肝素结合的影响。定点诱变和肝素结合数据表明精氨酸比 C 端赖氨酸对肝素结合过程至关重要，从而支持了这些 NMR 发现。
Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin, PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal a-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.