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Localizing and Modeling Headpiece Domains on F-Actin

F-肌动蛋白上头件结构域的本地化和建模

基本信息

批准号：
7026393
负责人：
Christopher James MCKNIGHT
金额：
$ 23.88万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-03-01 至 2007-02-28
项目状态：
已结题

项目摘要

With the exception the binding site of the C-terminal "headpiece" domain of villin, the structures and binding site sites of the actin crosslinking proteins that form the actin bundles that support the microvilli of the brush border epithelium have been identified and modeled. The headpiece domain is a modular, 76-amino acid F-actin binding motif that provides the essential second actin-binding site that allows villin to crosslink actin filaments into structural bundles. Headpiece domains are also found on at the C-termini of several classes of large "core" domains unrelated to villin. The focus of this proposal is on determining how and where headpiece domains bind actin filaments, to produce a model of the headpiece-actin complex, and to investigate the flexibility of sequence linking villin headpiece to its core domain. The broad, long-term objectives of this proposal are to determine at the molecular level how actin bundles like those that support the microvilli of brush border epithelial cells are assembled and organized. The specific aims of this proposal are: 1) To determine the high resolution structure of villin headpiece and the NMR structures the phosphoryl-regulated dematin headpiece, and the non-actin binding supervillin headpiece. 2) To test the published "hydrophobic cap, charged crown, and basic patch" hypothesis of headpiece-F-actin recognition by mutagenesis of the villin and supervillin headpiece domains 2. To locate the binding site of the headpiece motif on F-actin and computer models of the F-actin headpiece complex. 3. To determine the flexibility of the linkage between villin headpiece and its gelsolin- like core by 15N-NMR relaxation measurements. The health relatedness of this project is that by understanding how essential actin bundles, like those that support the absorptive epithelial brush border, are organized in the cell we may be able to manipulate their formation in epithelial and other cell types.

除了绒毛的 C 端“头”结构域的结合位点之外，形成支持刷状缘上皮微绒毛的肌动蛋白束的肌动蛋白交联蛋白的结构和结合位点已被识别和建模。头结构域是一个模块化的 76 个氨基酸 F-肌动蛋白结合基序，提供了重要的第二个肌动蛋白结合位点，使绒毛蛋白能够将肌动蛋白丝交联成结构束。头件结构域也存在于与villin无关的几类大“核心”结构域的C末端。该提案的重点是确定头件结构域如何以及在何处结合肌动蛋白丝，以生成头件-肌动蛋白复合物的模型，并研究将绒毛头件与其核心结构域连接的序列的灵活性。该提案的广泛、长期目标是在分子水平上确定肌动蛋白束（如支持刷状缘上皮细胞微绒毛的肌动蛋白束）如何组装和组织。该提案的具体目标是：1）确定绒毛头的高分辨率结构以及磷酰调节脱蛋白头和非肌动蛋白结合的超级绒毛头的NMR结构。 2) 通过绒毛和超绒毛头结构域的诱变来测试已发表的头-F-肌动蛋白识别的“疏水帽、带电冠和基本斑块”假说 2. 定位头部分基序在 F-肌动蛋白上的结合位点和 F-肌动蛋白头部分复合物的计算机模型。 3.通过15N-NMR弛豫测量来确定绒毛头件与其凝溶胶蛋白样核心之间的连接的灵活性。该项目与健康的相关性在于，通过了解必需的肌动蛋白束（如支持吸收性上皮刷状缘的肌动蛋白束）在细胞中的组织方式，我们也许能够操纵它们在上皮细胞和其他细胞类型中的形成。