PA63 channel of anthrax toxin:: An extended β-barrel
PA63 channel of anthrax toxin:: An extended β-barrel
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DOI：
10.1021/bi0119518
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发表时间：
2002-02-05
期刊：
影响因子：
2.9
通讯作者：
Finkelstein, A
中科院分区：
文献类型：
--
作者：
Nassi, S;Collier, RJ;Finkelstein, A
炭疽毒素由三种蛋白质组成：保护性抗原（PA）、致死因子（LF）和水肿因子（EF）。PA（63）由蛋白酶“切割”整个PA产生，负责将毒素的催化片段（LF和EF）递送至靶细胞的胞质溶胶。在平面双层膜中，胰蛋白酶切口的PA形成孔径大于或等于12埃的阳离子选择性电压门控通道。通道被推测为七聚体“蘑菇”，具有细胞外“帽”区域和插入膜的β-桶“茎”。虽然水溶性单体形式的晶体结构已解析到2.1埃，七聚体“前孔”的晶体结构解析到4.5埃，但膜结合通道（孔）的结构尚未确定。我们已经设计了突变体通道，其在假定的β-桶中的残基中被半胱氨酸取代，并通过其对水溶性巯基特异性试剂的可及性鉴定了通道内腔的残基。与腔暴露的半胱氨酰侧链的反应导致通道电导下降，我们用它来绘制排列在孔中的残基。我们的研究结果表明，β-桶结构延伸到双层之外，并涉及被埋在单体中的残基。这意味着，在prepore帽区域的结构域的主要重排是需要膜插入的β-桶茎。
Anthrax toxin consists of three protein components: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA(63), generated by protease "nicking" of whole PA, is responsible for delivering the toxin's catalytic fragments (LF and EF) to the target cell's cytosol. In planar bilayer membranes, trypsin-nicked PA makes cation-selective voltage-gated channels with a pore diameter of greater than or equal to 12 Angstrom. The channels are presumed to be heptameric "mushrooms", with an extracellular "cap" region and a membrane-inserted, beta-barrel "stem". Although the crystal structure of the water-soluble monomeric form has been resolved to 2.1 Angstrom and that of the heptameric "prepore" to 4.5 Angstrom, the structure for the membrane-bound channel (pore) has not been determined. We have engineered mutant channels that are cysteine-substituted in residues in the putative beta-barrel, and identified the residues lining the channel lumen by their accessibility to a water-soluble sulfhydryl-specific reagent. The reaction with lumen-exposed cysteinyl side chains causes a drop in channel conductance, which we used to map the residues that line the pore. Our results indicate that the beta-barrel structure extends beyond the bilayer and involves residues that are buried in the monomer. The implication is that major rearrangement of domains in the prepore cap region is required for membrane insertion of the beta-barrel stem.