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: 6622010
• 负责人: Christopher James MCKNIGHT
• 金额: $ 24.45万
• 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/6622010
• 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.

除了绒毛蛋白的C-末端“头部”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，提供了必要的第二肌动蛋白结合位点，允许绒毛交联肌动蛋白丝成结构束。在与绒毛蛋白无关的几类大的“核心”结构域的C-末端也发现了头片段结构域。这个建议的重点是确定如何和在哪里的头片段域结合肌动蛋白丝，产生一个模型的头片段-肌动蛋白复合物，并调查的灵活性连接绒毛蛋白头片段的核心结构域的序列。这个提议的广泛的，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，如那些支持刷状缘上皮细胞的微绒毛。本研究的具体目的是：1）确定绒毛蛋白头段的高分辨率结构和磷酸化调节的dematin头段和非肌动蛋白结合的超绒毛蛋白头段的NMR结构。2)通过对绒毛蛋白和超绒毛蛋白头部结构域的诱变来验证已发表的头部-F-肌动蛋白识别的“疏水帽、带电冠和基本补丁”假设2。确定F-肌动蛋白头片段基序的结合位点，并建立F-肌动蛋白头片段复合物的计算机模型。3.通过15 N-NMR弛豫测量来确定绒毛蛋白头部与其凝溶胶蛋白样核心之间的连接的柔性。该项目的健康相关性在于，通过了解基本肌动蛋白束（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，我们可能能够操纵它们在上皮和其他细胞类型中的形成。