Crystallography of Cholera and Heat Labile Enterotoxins

描述(申请人提供)：拟议的项目使用结晶学研究，以揭示两种细菌毒素，霍乱毒素(CT)和大肠杆菌不耐热肠毒素(LT)与一系列天然底物和相互作用的蛋白质，以及设计的配体和抑制剂的相互作用。CT引起的严重感染性腹泻可能导致几个小时内死亡，而产肠毒素LT的大肠杆菌引起的轻度腹泻主要是工业化国家居民的旅行者不适，但却是第三世界婴儿死亡的主要原因。CT和LT是非常复杂的异六聚体AB5毒素，它们具有许多有趣但尚未解决的特性，涉及它们进入宿主细胞的细胞质、它们的催化机制以及霍乱弧菌分泌CT的方式。根据上一个赠款期间的重大成果，我们建议在今后一段时期内，与几个具有互补专业知识的小组合作，处理结构研究，以阐明关于CT和LT的以下问题： 1.结合底物NAD和含精氨酸多肽的方式，以及ADP-核糖化A-亚基的酶机制，基于我们最近成功地确定了具有构成活性的CT的三维结构。这将包括一个刚刚启动的以结构为基础的药物设计合作项目，该项目采用了以胍为基础的新型化学物质，目的是阻断A亚单位； 2.CT和LT与宿主蛋白如ADP-核糖化因子(ARF)的相互作用； 3.设计超高亲和力的GM1受体拮抗剂的B-五聚体，采用多环系统的单价配体和新的“连接物”，以获得我们成功的下一代模块化五价和十价大分子配体； 4.霍乱弧菌胞外蛋白分泌装置中的EPSC的结构及其与CT的相互作用； 5.霍乱弧菌外膜多聚体孔蛋白EpsD的结构及其与EPSC的相互作用，以及可能与霍乱毒素的相互作用。

DESCRIPTION (provided by applicant): The proposed project uses crystallographic studies to unravel the interactions of two bacterial toxins, cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT), with a range of natural substrates and interacting proteins, as well as with designed ligands and inhibitors. The severe infectious diarrhea caused by CT may result in death within a few hours, while the milder diarrhea caused by enterotoxigenic LT-producing E. coli is mainly a traveler's discomfort for inhabitants of industrialized countries, but a major cause of infant death in the third world. CT and LT are remarkably sophisticated hetero-hexameric AB5 toxins that have many intriguing yet unresolved properties concerning their entry into the cytoplasm of the host cell, their catalytic mechanism, and the mode of secretion of CT from Vibrio cholerae. Based on significant results in the previous grant period we propose in the coming period to tackle, in collaboration with several groups with complementary expertise, structural studies to elucidate the following regarding CT and LT: 1. the mode of binding the substrates NAD and arginine-containing peptides, and the enzymatic mechanism of the ADP-ribosylating A-subunit, based on our recent success in determining the three-dimensional structure of a constitutively active form of CT. This will include a just initiated structure-based drug design collaborative project employing novel guanidine-based chemistry aimed at blocking the A-subunit; 2. the interactions of CT and LT with host proteins such as ADP-Ribosylation Factors (ARFs); 3. the design of ultra-high affinity GM1 receptor antagonists of the B-pentamer by employing multi-ring system-containing monovalent ligands and new "linkers" to obtain a next generation of our successful modular pentavalent and decavalent macroligands; 4. the structure of EpsC, a periplasmic inner membrane-anchored protein from the extracellular protein secretion apparatus in V. cholerae, and its interactions with CT; 5. the structure of the multimeric pore protein EpsD in the outer membrane of V. cholerae and its interactions with EpsC, and possibly, with cholera toxin.