Full-contact domain labeling: Identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein
Full-contact domain labeling: Identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein
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DOI：
10.1021/bi000996q
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发表时间：
2001-01-30
期刊：
影响因子：
2.9
通讯作者：
Prestwich, GD
中科院分区：
文献类型：
--
作者：
Feng, L;Mejillano, M;Prestwich, GD
凝溶胶蛋白是一种肌动蛋白和磷酸肌醇结合蛋白，使用多种含二苯甲酮的磷酸肌醇多磷酸类似物进行光亲和标记。凝溶胶蛋白的 N 端一半和 C 端一半在磷脂酰肌醇 3,5-二磷酸 [PtdIns(4,5)P-2] 的结合中显示出协同作用。 PtdIns(4,5)Pz 的二丁酰基、二辛酰基或二棕榈酰基衍生物的竞争性置换实验表明，除了肌醇头基之外，二酰基甘油基部分对于结合也很重要；这些类似物还抑制 F-肌动蛋白的凝溶胶蛋白切割活性。除了之前在凝溶胶蛋白 N 端一半中鉴定的 PtdIns(4,5)Pz 结合位点外，通过对光共价修饰的肽片段进行绘图，在 C 端一半中鉴定了一个新的结合位点。此外，Ca2+浓度的增加降低了光不稳定类似物与凝溶胶蛋白上C端磷酸肌醇结合位点的结合。使用这些数据计算了凝溶胶蛋白两个折叠重复内的 nf PtdIns (4,5)P-2 结合的分子模型。
Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polyphosphate analogues. The N-terminal half and he C-terminal half of gelsolin showed synergy in the binding of phosphatidylinositol 3,5-bisphosphate [PtdIns(4,5)P-2]. Competitive displacement experiments with dibutyryl, dioctanoyl, or dipalmitoyl derivatives of PtdIns(4,5)Pz suggested that, in addition to the inositol headgroup, a diacylglyceryl moiety was important for binding; these analogues also inhibited the gelsolin-severing activity of F-actin. In addition to the previously identified PtdIns(4,5)Pz binding site in the N-terminal half of gelsolin, a new binding site was identified in the C-terminal half by mapping the photocovalently modified peptide fragments. Moreover, increasing concentrations of Ca2+ decreased the binding of the photolabile analogues to the C-terminal phosphoinositide binding site on gelsolin. A molecular model of the binding nf PtdIns (4,5)P-2 within two folded repeats of gelsolin has been calculated using these data.