Follistatin-Molecular Structure/Function

卵泡抑素-分子结构/功能

• 批准号: 7193445
• 负责人: HENRY T KEUTMANN
• 金额: $ 25.55万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7193445
• 关键词: Activin Receptor; Activins; Amino Acids; Architecture; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Cell surface; Cells; Characteristics; Chemicals; Complement; Complex; Crystallization; Crystallography; Cultured Cells; Cysteine; Development; Evaluation; Extracellular Protein; Family; Follistatin; Future; Goals; Growth Factor; Heparan Sulfate Proteoglycan; Heparin Binding; Hydrophobic Surfaces; Investigation; Length; Ligands; Maintenance; Mediator of activation protein; Modeling; Modification; Molecular; Molecular Conformation; Molecular Structure; Mutagenesis; N Domain; N-terminal; Ovarian; Physiological; Pituitary Gland; Protein Binding; Range; Regulation; Resolution; Role; Serum-Free Culture Media; Site; Staging; Structure; Surface; Suspension Culture; System; Testing; Tissues; Transforming Growth Factor beta; Work; X ray diffraction analysis; X-Ray Diffraction; activin A; autocrine; base; bone; chordin; insight; mutant; receptor binding; reproductive; three dimensional structure; three-dimensional modeling

DESCRIPTION (provided by applicant): The goal of this project has been to establish the role of domain structure and determine the structural requirements for the biological activity of follistatin, a multidomain protein that binds and bioneutralizes activin in pituitary, bone and a wide range of other tissues and physiological systems. Our investigations to date of the 288-residue follistatin molecule have concentrated on functional effects of chemical modifications and mutagenesis on activin binding, as well as on association with cell-surface heparan-sulfate proteoglycans that promote follistatin's tissue localization. These have shown that the 63-residue N-terminal domain plus the first two of the three distinctive 10-cysteine "follistatin" (FS) domains are required for activin binding, and that hydrophobic residues are particularly important both to activin interaction and maintenance of domain conformation. Under this renewal, we will use complementary approaches of crystallography and mutational analysis to examine interdomain relationships and the organization of activin-binding components to provide a functional model of the follistatin molecule. Under Aim 1 we will determine the 3-dimensional structure of the purified follistatin-288 molecule by crystallization and x-ray diffraction analysis. Structures will be done on full-length follistatin and (if needed to enhance resolution) two-domain segments, purified from 293F-cell suspension cultures in serum-free medium. In Aim 2 we will prepare follistatin complexed with activin for crystallography to directly identify contact sites and document changes undergone by key determinants during complex formation. Mutational analyses of the activin ligand under Aim 3 will complement and functionally validate the structures developed from crystallographic analysis of the activin-follistatin complex. Identification of functionally important hydrophobic surface residues will test our hypothesis that follistatin acts through competition with known hydrophobic sites in the activin receptor. These investigations will clarify the mechanism of the widely-postulated local regulation of activin by follistatin, enhance understanding of the activity of other potent binding proteins (such as noggin and chordin) characteristic of the TGF-beta family and expand our insight into the role of follistatin-domain structures in general among the many other extracellular proteins in which they can be found.

描述（由申请人提供）：本项目的目标是确定结构域结构的作用，并确定卵泡抑素生物活性的结构要求，卵泡抑素是一种多结构域蛋白，可结合垂体、骨和广泛的其他组织和生理系统中的激活素并对其进行生物中和。迄今为止，我们对288个残基的卵泡抑素分子的研究主要集中在化学修饰和诱变对激活素结合的功能影响，以及与促进卵泡抑素组织定位的细胞表面硫酸乙酰肝素蛋白聚糖的相关性。这些已经表明，63个残基的N-末端结构域加上三个独特的10-半胱氨酸“卵泡抑素”（FS）结构域中的前两个是激活素结合所需的，并且疏水残基对于激活素相互作用和结构域构象的维持都特别重要。在此更新下，我们将使用结晶学和突变分析的互补方法来研究结构域间的关系和激活素结合组分的组织，以提供卵泡抑素分子的功能模型。在目标1下，我们将通过结晶和X射线衍射分析确定纯化的卵泡抑素-288分子的三维结构。将对全长卵泡抑素和（如果需要提高分辨率）从无血清培养基中的293 F细胞悬浮培养物中纯化的双结构域片段进行结构分析。在目标2中，我们将制备卵泡抑素与激活素的晶体学复合物，以直接识别接触位点并记录复合物形成过程中关键决定因素的变化。目标3下激活素配体的突变分析将补充和功能验证从激活素-卵泡抑素复合物的晶体学分析开发的结构。功能上重要的疏水表面残基的鉴定将测试我们的假设，卵泡抑素的行为，通过与已知的疏水位点的激活素受体的竞争。这些调查将澄清广泛假设的局部调节激活素卵泡抑素的机制，增强对其他有效的结合蛋白（如头蛋白和腱蛋白）的TGF-β家族的特征的活性的理解，并扩大我们的洞察力卵泡抑素结构域的作用，一般在许多其他细胞外蛋白，其中可以找到他们。