Protein Unfolding During Anthrax Toxin Translocation

描述(由申请人提供)： 细菌病原体已经进化出一种通过形成AIB机制的毒素蛋白复合体来感染宿主生物的一般策略。活性的、酶促的A部分与受体B部分结合；然后，受体部分充当载体，将A部分从由膜双层隔开的孤立的隔间输送到细胞质。然后，酶效应器通过其特定的催化活性，使细胞的正常生理功能失效。炭疽芽孢杆菌的三体毒素依赖于一种“分子注射器”B部分，即保护性抗原(PA)，在中性的胞外pH条件下，PA形成具有狭窄中央空腔的七元环结构。两个不同的A部分效应物，致死因子(Lf)和水肿因子(EF)，结合PA七聚体。膜结合的PA/LF/EF复合体内吞后立即发生酸化，触发PA七聚体的膜穿透孔形式的同步形成，推测该七聚体将LF和EF注入细胞质。PA七聚体的狭窄中央空腔太小，无法容纳完全天然的LF或EF，这意味着蛋白质的展开可以减轻易位的可能性。事实上，LF的稳定化融合验证了这种机制。各种荧光光谱和成像方法的现代进步使人们能够在单分子水平上详细地阐明蛋白质的折叠途径。使用荧光方法对导致和紧随易位的事件序列进行彻底的结构、能量和动力学理解，将有助于改进对设计和实施潜在的中毒治疗方法的讨论，包括设计的蛋白质抑制剂和药物。

DESCRIPTION (provided by applicant): Bacterial pathogens have evolved a general strategy of infecting host organisms by means of toxin protein complexes that pattern the AIB mechanism. The active, enzymatic, A moiety binds the receptor, B moiety; the receptor moiety then acts as a vehicle that delivers the A moiety to the cytoplasm from an isolated compartment separated by a membrane bilayer. The enzymatic effector, by means of its specific catalytic activity, then disables the normal physiology of the cell. The tripartite toxin of Bacillus anthracis relies on a "molecular syringe" B moiety, Protective Antigen (PA), which forms a heptad ring structure with a narrow central cavity at neutral extracellular pH. Two different A moiety effectors, Lethal Factor (LF) and Edema Factor (EF), bind PA heptamer. Immediately following endocytosis of membrane bound PA/LF/EF complexes, endosomes acidify, triggering the synchronized formation of a membrane piercing pore form of PA heptamer that presumably injects LF and EF into the cytosol. The narrow central cavity of PA heptamer is too small to accommodate fully native LF or EF, invoking the possibility that translocation is mitigated by protein unfolding. Indeed, stabilized fusions of LF validate this type of mechanism. Modern advances in a variety of fluorescence spectroscopy and imaging methods have enabled the detailed elucidation of protein folding pathways even at the single molecule level. A thorough structural, energetic, and kinetic understanding of the sequence of events leading up to and immediately following translocation using fluorescence methods will enable improved discourse toward the design and implementation of potential therapies for intoxication, including designed protein inhibitors and drugs.