DIFFRACTION OF BACTERIAL ENDOTOXINS: STRUCT BASED DRUG DESIGN, SCREEN PCP ENZYME

细菌内毒素的衍射：基于结构的药物设计，筛选 PCP 酶

• 批准号: 6220476
• 负责人: Vivian Cody
• 金额: $ 1.38万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-15 至 2000-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6220476
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

Delta endotoxins are the entomocidal agent produced by Bacillus thuringiensis ssp. Kurstaki. These toxins are produced as protoxins (135 kDa) by the bacteria during sporulation and recognized by their bipyrimidal crystalline form in the sporangium. When ingested by larval Lepidoptera the crystal dissolves and is proteolytically digested to the active toxin form (60-65 kDa). This activated toxin is capable of opening K+ channels in the epithelium of the insect midgut. Ion leakage ultimately results in the complete disruption of the midgut and death of the insect. More recently, a new active form of the toxin, CryIIIB2, has been determined (64 kDa). The bacterial endotoxin CryIIIB2 is a 652 residue protein which crystallizes in the orthorhombic space group C2221 with unit cell dimensions a = 122.44, b = 131.81, and c = 105.37& and contain one molecule in the asymmetric unit. Synchrontron radiation was used to collect 2.2& resolution data at the CHESS facility at Cornell University in December, 1997 by Vivian Cody and Nikolai Galitsky for the complex of EC11095 with D-galactosamine. Although these data were did not diffract uniformly to high resolution, Walt Pangborn was able to scale data with at least 50% completion for all shells to 2.2& resolution. The structure of the native CryIIIB2 was used as a search model for the rotation and translation function in the program XPLOR which revealed there was no significant change in the molecular orientation of the molecule compared to the room temperature model. The major consequence of low temperature data collection is the contraction of the unit cell along the a and b lattice directions. Interpretation of the difference electron density maps made from the 3.0& refinement model of the Arg-348 mutant of CryIIIB2 revealed a large density profile that clearly was not a cluster of solvent and that could be fit to the sugar. These data reveal that the galactosamine is positioned at the intersection of the three domains of CryIIIB2. Refinement of these data to 2.2& resolution suggest other potential sugar binding sites are possible.

Delta内毒素是芽孢杆菌产生的杀虫物质 苏云金杆菌SSP.库尔斯塔基。这些毒素以原毒素的形式产生 (135 KDa)被细菌在产孢子过程中识别并被它们的 孢子囊中的双锥体晶形。当被摄取时 鳞翅目幼虫晶体溶解并蛋白降解 被消化成活性毒素形式(60-65 kDa)。这种被激活的毒素 能够打开昆虫上皮中的K+通道 中肠。离子泄漏最终会导致核辐射的完全破坏 昆虫的中肠和死亡。最近，一种新的活动表单 已确定该毒素为CryIIIB2(KDa)。细菌 内毒素CryIIIB2是一种由652个残基组成的蛋白质。 晶胞尺寸a=122.44，b的正交空间群C2221 =131.81，c=105.37，并且包含一个不对称分子 单位。使用同步辐射收集2.2分辨率的数据 1997年12月在康奈尔大学的国际象棋设施被 维维安·科迪和尼古拉·加利茨基为EC11095和 D-氨基半乳糖。尽管这些数据并不均匀地衍射化 为了达到高分辨率，沃尔特·庞伯恩能够用至少 完成50%的所有炮弹到2.2和分辨率。的结构 使用本地的CryIIIB2作为旋转和 程序XploR中的翻译功能显示没有 分子的分子取向发生重大变化 与室温模型进行了比较。Low的主要后果 温度数据采集是单位电池沿距离的收缩 A和b晶格方向。对差异的解释 从3.0版制作的电子密度图&精化模型 CryIIIB2的Arg-348突变体显示出较大的密度分布 显然不是一簇溶剂，这可以适用于 糖。这些数据表明，氨基半乳糖位于 CryIIIB2的三个结构域的交集。对这些进行改进 到2.2和分辨率的数据表明了其他潜在的糖结合位点 都是可能的。