Follistatin-Molecular Structure/Function

描述(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质，可结合和生物中和脑垂体、骨骼和广泛的其他组织和生理系统中的激活素。到目前为止，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突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-表面硫酸肝素蛋白多糖的结合。这些研究表明，63个残基的N末端结构域加上三个不同的10-半胱氨酸“卵泡抑素”(FS)结构域中的前两个结构域是激活素结合所必需的，并且疏水残基对激活素相互作用和结构域构象的维持都特别重要。在这次更新中，我们将使用结晶学和突变分析的互补方法来检查结构域之间的关系和激活素结合组件的组织，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。结构将在全长卵泡抑素和(如果需要提高分辨率)两个结构域片段上完成，这些片段是从293F细胞悬浮培养物中在无血清介质中纯化的。在目标2中，我们将制备卵泡抑素与激活素的复合体，用于结晶学，以直接确定接触点并记录在复合体形成过程中关键决定因素所经历的变化。根据目标3对激活素配体的突变分析将补充并在功能上验证从激活素-卵泡抑素复合体的结晶学分析中发展出来的结构。对功能上重要的疏水表面残基的鉴定将检验我们的假设，即卵泡抑素通过与激活素受体中已知的疏水位点竞争而发挥作用。这些研究将阐明广泛假设的卵泡抑素对激活素的局部调节机制，增进对转化生长因子-β家族其他有效结合蛋白(如noggin和chordin)活性的了解，并扩大我们对卵泡抑素结构域在许多其他胞外蛋白中的作用的认识。

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.