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

• 批准号: 6438240
• 负责人: Christopher James MCKNIGHT
• 金额: $ 25.27万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/6438240
• 关键词: X ray crystallography; actin binding protein; actins; adenosine triphosphate; computer simulation; conformation; crosslink; crystallization; high performance liquid chromatography; image processing; intermolecular interaction; membrane proteins; microfilaments; model design /development; molecular assembly /self assembly; molecular site; nuclear magnetic resonance spectroscopy; phosphorylation; physical model; protein purification; protein sequence; protein structure function; radiotracer; site directed mutagenesis; structural biology

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villin的C末端“头饰”结构域的结合位点，肌动蛋白交联蛋白的结构和结合位点已鉴定并建模，这些蛋白质形成了支持刷子边框上皮的微维利利的肌动蛋白捆绑。头式结构域是一个模块化的76个氨基酸F-肌动蛋白结合基序，提供了必不可少的第二肌动蛋白结合位点，使Villin可以交叉链接肌动蛋白丝进入结构束。在C-termini上还发现了头饰域，该类别的几类与Villin无关的大型“核心”域。该提案的重点是确定头型结构域如何和何处结合肌动蛋白丝，以产生头骨 - 肌动蛋白复合物的模型，并研究将villin头饰与其核心结构域连接的序列的灵活性。该建议的广泛，长期的目标是在分子水平上确定肌动蛋白束如何组装和组织刷子边界上皮细胞的微维利。该提案的具体目的是：1）确定villin头饰的高分辨率结构和NMR结构的磷酸化调节的dematin头饰，以及非肌动蛋白结合的SupperIllin头饰。 2）通过测试已发表的“疏水帽，带电的冠状和基本斑块”假设，该假设是通过对villin和supervillin头上头的诱变来测试头寸F-肌动蛋白识别的假设。以定位F-actin和F-Actin Headpiese配合物的F-actin和计算机模型的头饰图案的结合位点。 3。确定villin头饰与其凝胶蛋白像核心之间的连接性的灵活性，通过15N-NMR松弛测量。该项目的健康相关性是，通过了解基本的肌动蛋白捆绑包（例如支持吸收性上皮刷边界的基本捆绑包）如何在细胞中组织起来，我们可能能够在上皮和其他细胞类型中操纵它们的形成。