Blood Coagulation Protein-Metal Ion-Lipid Interactions

描述（由申请人提供）：本研究计划的总体目标是在超过30年的时间内确定体外和体内维生素K依赖性凝血蛋白的结构功能关系，特别关注γ-羧基谷氨酸（Gla）结构域（GD）与金属离子、膜和受体的相互作用。在这段时间里，我们已经解决了这些问题，通过各种生物物理技术，蛋白质化学，分子/细胞生物学，和基因靶向策略。 在当前的更新申请中，将集中精力研究这些含Gla的肽的结构、动力学和功能，特别关注蛋白C（PC）/内皮细胞蛋白C受体（EPCR）系统和含Gla的伴体蛋白，这是凝血蛋白GD的神经活性模型。提出了三个相互关联的具体目标： (1)为了化学合成特定的含Gla的芋螺毒素，即，conG、conT、conR（1-17）和conL，以及这些肽的关键变体，以及：（a）采用电生理学和[3 H] MK 801结合来定量它们对由NR 1和NR 2亚基的已知组合组成的重组NMDA受体（NMDAR）的抑制效力，并描绘涉及它们的NMDAR亚基特异性的特定残基;（B）克隆和表达编码NMDAR的NR 2 B亚基的非邻接Glu结合结构域的重组构建体，并更充分地评估凝固因子Glu对该受体区段的伴体蛋白抑制的性质;（c）鉴定参与其与NMDAR的NR 2B亚基结合的conR残基，并使用同位素-NMR上的多维异源和同源NMR研究conR（1-17）与其NMDAR受体片段结合时的溶液结构。富集conR和受体;（d）研究在Ca 2+存在下，conG所采用的独特的金属离子诱导的GD二聚体超结构的稳定作用。 2)表达鼠可溶性重组全氘代[2 H/13 C/15 N]-EPCR，并测定脂化sr-EPCR + GDPC的骨架分子动力学（1-47）。 3)化学合成小鼠PC的GD（GDPC 1 -47），沿着与含有单个氨基酸置换的类似物，以及：（a）使用生物物理学方法检查这些结构域与Ca 2+、Mg 2+和Zn 2+以及与鼠sr-EPCR的相互作用;和（B）用NMR和X-射线晶体学确定钙和镁复合的鼠GDPC（1-47）的溶液和晶体结构以及主链动力学。 这些目标的实现将有助于我们理解这些GD的关键结构特征，这些GD允许含有这些区域的蛋白质与其受体的特异性功能结合。

DESCRIPTION (provided by applicant): The overall goals of this research program over a more than a 30-year period are to define structure function relationships of vitamin K-dependent coagulation proteins, both in vitro and in vivo, with specific attention paid to interactions of the gamma-carboxyglutamic acid (Gla) domains (GD) with metal ions, membranes, and receptors. Over this period of time, we have addressed these issues by a variety of biophysical techniques, and by protein chemistry, molecular/cell biology, and gene targeting strategies. In the current renewal application, efforts will be concentrated on the structures, dynamics, and functions of these Gla-containing peptides with specific attention on the Protein C (PC)/Endothelial Cell Protein C Receptor (EPCR) system, and on Gla-containing conantokins, which are neuroactive models of GDs of coagulation proteins. Three interrelated specific aims are proposed: (1) To chemically synthesize specific Gla-containing conantokins, viz., conG, conT, conR (1-17), and conL, as well as strategic variants of these peptides, and: (a) to employ electrophysiology and [3H]MK801 binding to quantitate their inhibitory potency toward recombinant NMDA receptors (NMDAR) comprised of known combinations of NR1 and NR2 subunits and to delineate the specific residues involved in their NMDAR subunit specificity; (b) to clone and express a recombinant construct encoding the noncontiguous Glu-binding domains of the NR2B subunit of the NMDAR and to more fully assess the nature of the inhibition of the conantokins with the coagonist, Glu, on this receptor segment; (c) to identify conR residues involved in its binding to the NR2B subunit of the NMDAR and to study the solution structure of conR (1-17) when bound to its NMDAR receptor segment using multidimensional hetero- and homonuclear NMR on isotopically-enriched conR and receptor; and (d) to study the interactions that stabilize the unique metal ion-induced GD dimeric superstructure that is adopted by conG in the presence of Ca 2+. 2) to express murine soluble (s) recombinant (r) perdeuterated [2H/13C/15N]-EPCR and to determine backbone molecular dynamics of lipidated sr-EPCR + GDPC (1-47). 3) To chemically synthesize the GD of murine PC (GDPC1-47), along with analogs containing individual amino acid replacements, and: (a) to examine the interactions of these domains with Ca 2+, Mg 2+, and Zn 2+, and with murine sr-EPCR, using biophysical methods; and (b) to determine the solution and crystal structures and backbone dynamics of Ca 2+ - and Mg 2+ -complexed murine GDPC (1-47) using NMR and X-ray crystallography. Accomplishment of these goals will assist in our understanding of the critical structural features of these GDs that allow the specific functional binding of proteins containing these regions to their receptors.