ANNEXIN CRYSTAL STRUCTURES AND MEMBRANE INTERACTIONS

在细胞膜-胞质溶胶界面处有活性的蛋白质以一种 独特的两相环境，其分子机制代表了 细胞生物学的前沿领域 膜联蛋白是一个家族的界面 钙依赖性膜结合蛋白，广泛分布于 真核生物 在许多细胞中发现非常大量（1-2%总细胞蛋白） 在组织中，膜联蛋白以水溶性和膜结合形式稳定存在。 对于结构生物学家来说，膜联蛋白提供了一个不寻常的研究机会， 无数的蛋白质介导的膜过程。 膜联蛋白V，一种假定的 钙跨膜运动的效应，提供了一个很好的 用于研究膜联蛋白家族和其他界面的结构原型 proteins. 这项研究计划的长期目标是开发一种 膜联蛋白在膜上行为的综合图。 首要目标 提出的研究是开发一种膜结合的分子模型， 膜联蛋白V，详细描述了它与膜组分的相互作用， 表征其对膜参数的影响。 x射线晶体学和 解决方法将被用作研究这两个问题的补充方法 水溶性和膜结合形式的膜联蛋白V.的具体目的 本研究拟：1）膜联蛋白配体的晶体学研究 相互作用; 1a）用钙，脂质， 和磷脂类似物结合; 1b）确定 膜联蛋白V复合物与一系列脱酰或短链 磷脂酰丝氨酸化合物; 1c）解决非磷脂酰丝氨酸化合物的结构 与膜联蛋白V中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复合物的结构。 （二） 研究膜联蛋白V的性质诱变，使用晶体学和 溶液分析（蛋白质-囊泡结合、化学交联、NMR 光谱法，呋喃-2-荧光测定法）：2a）在非- 功能性钙结合环，以观察钙结合是否可以恢复; 2b）E产生V突变，导致在其他情况下的钙结合丧失， 功能环，以观察对磷脂头部基团的影响 在这个位点结合; 2c）替换推定的决定簇， 膜联蛋白V的脂质结合偏好是否可以改变为 膜联蛋白I，以探测磷脂特异性的结构基础; 2d） 破坏三聚体形成的突变，以研究 寡聚化和其它膜联蛋白性质。 3)结晶，以进一步 3a）磷酸化的膜联蛋白iV，以研究膜联蛋白iV的磷酸化。 这种修饰的结构后果;和3b）膜联蛋白Xi，其， 与膜联蛋白V不同，它具有广泛的N-末端结构域。 信息从 这些研究将大大增加相当稀疏的知识， 界面蛋白为膜联蛋白提供了统一的结构基础 作用于膜，并提出了推定的结构基础 膜联蛋白V功能可能与肾、肺、肝和其它器官有关 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.