Localizing and Modeling Headpiece Domains on F-Actin

除了绒毛蛋白c端“头套”结构域的结合位点外，形成支持刷状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经被鉴定和建模。头部结构域是一个模块化的76个氨基酸的f -肌动蛋白结合基序，它提供了必不可少的第二个肌动蛋白结合位点，允许绒毛蛋白将肌动蛋白丝交联成结构束。在与绒毛蛋白无关的几类大型“核心”结构域的c端也发现了头部结构域。本提案的重点是确定头饰结构域与肌动蛋白丝结合的方式和位置，产生头饰-肌动蛋白复合物的模型，并研究将绒毛蛋白头饰与其核心结构域连接的序列的灵活性。本研究的长远目标是在分子水平上确定支持刷状上皮细胞微绒毛的肌动蛋白束是如何组装和组织的。本课题的具体目的是：1)确定高分辨率绒毛蛋白头套的结构和磷酸化调控的蛋白头套、非肌动蛋白结合的超级绒毛蛋白头套的核磁共振结构。2)验证已发表的通过诱变绒毛蛋白和超级绒毛蛋白头饰结构域识别头饰- f -actin的“疏水帽、带电冠和基本贴片”假说。定位头饰基序在F-actin上的结合位点和F-actin头饰复合物的计算机模型。3. 通过15N-NMR弛豫测量来确定绒毛头与胶状芯之间的连接的柔韧性。这个项目与健康的关系是，通过了解基本的肌动蛋白束，比如那些支持吸收性上皮刷状边界的肌动蛋白束，在细胞中是如何组织的，我们可能能够操纵它们在上皮细胞和其他细胞类型中的形成。

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.