Localizing and Modeling Headpiece Domains on F-Actin

除了绒毛蛋白C-末端“头饰”结构域的结合位点外，肌动蛋白交联蛋白的结构和结合位点已经被识别和建模，这些蛋白形成了支持刷状边缘上皮微绒毛的肌动蛋白束。头戴结构域是一个由76个氨基酸组成的模块化的F-肌动蛋白结合基序，它提供了必要的第二个肌动蛋白结合位点，使Villin能够将肌动蛋白细丝交联成结构束。头戴式结构域也在几类与Villin无关的大的“核心”结构域的C-末端被发现。这项建议的重点是确定头盔结构域如何以及在哪里结合肌动蛋白细丝，建立头盔-肌动蛋白复合体的模型，并研究将Villin头饰与其核心域连接的序列的灵活性。这项提议的广泛、长期的目标是在分子水平上确定肌动蛋白束是如何组装和组织的，比如那些支持刷状边缘上皮细胞微绒毛的肌动蛋白束。这项建议的具体目的是：1)确定绒毛蛋白头盔的高分辨结构和核磁共振结构，包括磷酸化调控的Dematin头盔和非肌动蛋白结合的超级绒毛头盔。2)通过对F-肌动蛋白和超绒毛蛋白头盔结构域的突变，验证已发表的关于头盔-F-肌动蛋白识别的“疏水帽、带电冠和基本斑块”假说。2.定位头盔基序在F-肌动蛋白上的结合位置和F-肌动蛋白复合体的计算机模型。3.通过~(15)N-核磁共振松弛测量确定维林头盔与其凝胶样芯之间连接的柔韧性。这个项目与健康相关的是，通过了解基本的肌动蛋白束是如何在细胞中组织起来的，比如那些支持吸收上皮刷状边界的肌动蛋白束，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.