ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS

在细胞膜-细胞质界面上活跃的蛋白质在 独特的两相环境及其分子机制代表着一种 当前细胞生物学的前沿。膜联蛋白是一个界面蛋白家族 钙依赖的膜结合蛋白广泛分布于 真核生物。发现大量的(1-2%的细胞总蛋白)在许多 在组织中，膜联蛋白以水溶和膜结合的形式稳定存在。 对于结构生物学家来说，膜联蛋白提供了一个不同寻常的研究机会 无数的蛋白质介导的膜过程。Annexin V，一个推定的 钙跨膜移动的效应器，提供了一种极好的 用于研究膜联蛋白家族和其他界面的结构原型 蛋白质。这项研究计划的长期目标是开发一种 膜上膜联蛋白行为的综合图像。首要目标 建议的研究内容之一是开发膜结合的分子模型 Annexin V，详细说明其与膜组件的相互作用，以及 表征其对膜参数的影响。X射线结晶学和 解决方法将被用作研究两者的互补方法 膜联蛋白V的水溶性和膜结合型。 这一建议是为了：1)用结晶学方法研究膜联蛋白配体 相互作用；1a)与钙、脂类、 和磷脂类似物结合；1b)确定 具有一系列脱酰化或短链的膜联蛋白V络合物 磷脂酰丝氨酸化合物；1c)解决非磷脂结构 与Annexin V分子间疏水位点结合的化合物 晶体；1D)确定碳水化合物-膜联蛋白V复合体的结构。2) 通过突变研究膜联蛋白V的性质，使用结晶学和 溶液分析(蛋白质-囊泡结合、化学交联、核磁共振 光谱学，Fura-2-荧光测定法)：2A)A K在非 有功能的钙结合环，看看钙结合能否恢复； 2B)E产生V突变，导致钙结合丢失 功能环，以查看结果对磷脂头部基团的影响 在这个位点结合；2c)假定决定因素的替代，见 膜联蛋白V的脂质结合偏好能否改变为 膜联蛋白I，探讨磷脂专一性的结构基础；2D) 破坏三聚体形成的突变，来研究它们之间的关系 齐聚和其他膜联蛋白特性。3)结晶，以便进一步 研究，另外两个膜联蛋白；3a)磷酸化的膜联蛋白IV，以研究 此修改的结构后果；以及3b)Annexin XI， 与Annexin V不同的是，它具有广泛的N-末端结构域。信息来自 这些研究将大大增加相当稀少的关于 界面蛋白，为膜联蛋白提供统一的结构基础 在膜上的作用，并提出了推测的结构基础 膜联蛋白V的功能可能与肾、肺、肝等器官有关 健康和疾病中的组织。

Proteins that are active at cell membrane-cytosol interfaces operate in a unique, two-phase environment, and their molecular mechanisms represent a current frontier of cell biology. Annexins are a family of interfacial calcium-dependent membrane-binding proteins that are widely distributed in eukaryotes. Found in very large amounts (1-2% total cell protein) in many tissues, annexins exist stably in water-soluble and membrane-bound forms. For structural biologists, annexins present an unusual opportunity to study a myriad of protein-mediated membrane processes. Annexin V, a putative effector of calcium movement across membranes, has provided an excellent structural prototype for study of the annexin family and other interfacial proteins. The long-term goal of this research program is to develop an integrated picture of annexin behavior at the membrane. The primary goal of the proposed research is to develop a molecular model of membrane-bound annexin V, detail its interactions with membrane components, and characterize its effects on membrane parameters. X-ray crystallography and solution methods will be used as complementary approaches to study both water-soluble and membrane-bound forms of annexin V. The specific aims of this proposal are to; 1) Crystallographically study annexin-ligand interactions; 1 a) Refine solved crystal structures with calcium, lipid, and phospholipid analogues bound; 1b) determine crystal structures of annexin V complexes with a series of deacylated or short-chain phosphatidylserine compounds; 1 c) solve structures of non-phospholipid compounds bound to an intermolecular hydrophobic site in annexin V crystals; 1d) determine structures of carbohydrate-annexin V complexes. 2) Investigate annexin V properties by mutagenesis, using crystallography and solution assays (protein-vesicle binding, chemical cross-linking, NMR spectroscopy, fura-2-fluorimetry): 2a) A K yields E mutation in a non- functional calcium-binding loop to see if calcium-binding can be restored; 2b) an E yields V mutation causing loss of calcium binding in an otherwise functional loop, to see the resulting effect on phospholipid head-group binding at this site; 2c) substitution of putative determinants to see whether the lipid binding preference of annexin V can be changes to that of annexin I, to probe the structural basis of phospholipid specificity; 2d) mutations that disrupt trimer formation, to study the relationship between oligomerization and other annexin properties. 3) Crystallize, for further study, two other annexins; 3a) phosphorylated annexin iV, to study the structural consequences of this modification; and 3b) annexin XI, which, unlike annexin V, has an extensive N-terminal domain. Information from these studies will add much to the rather sparse body of knowledge about interfacial proteins, provide a unifying structural basis for annexin action at he membrane, and propose the structural basis for putative annexin V function as it may relate to kidney, lung, liver, and other organ tissues in health and disease.