Anthrax protective antigen: Prepore-to-pore conversion
Anthrax protective antigen: Prepore-to-pore conversion
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DOI：
10.1021/bi990792d
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发表时间：
1999-08-10
期刊：
影响因子：
2.9
通讯作者：
Collier, RJ
中科院分区：
文献类型：
--
作者：
Miller, CJ;Elliott, JL;Collier, RJ
PA(63) 是炭疽保护性抗原的活性 63 kDa 形式，形成七聚环状寡聚物，据信该寡聚物代表了由该蛋白质形成的膜孔的前体。当 pH 值保持在大于或等于 8.0 时，这种“前孔”在室温下用 SDS 处理后解离为单体亚基，但在 pH 值小于或等于 7（或用 β-辛基葡萄糖苷在 pH 8.0 下处理）时，会导致其转化为 SDS 抗性孔状形式。向这种形式的转变涉及结构域 2 (D2L2) 的环 2 构象的重大变化，如 (i) D2L2 内胰凝乳蛋白酶位点的闭塞和 (ii) 与该环内的 N306C 连接的芘基团形成准分子所证明。孔状形式保留了结合炭疽毒素 A 部分和细胞表面受体的能力，但无法在膜中形成孔或介导易位。 D2L2 被删除的突变体 PA(63) 在孔形成和易位方面没有活性，但与前孔一样，能够形成七聚体，并在酸性条件下转化为 SDS 抗性形式。我们的研究结果支持孔形成模型，其中 D2L2 环移动到七聚体的膜近端面并相互作用形成 14 链跨膜 β 桶。与此同时，结构域 2 经历了独立于 D2L2 的主要构象重排，这使得七聚体能够抵抗 SDS 的解离。这些结果为进一步探索PA(63)在炭疽毒素酶基跨膜易位中的作用提供了基础。
PA(63), the active 63 kDa form of anthrax protective antigen, forms a heptameric ring-shaped oligomer that is believed to represent a precursor of the membrane pore formed by this protein. When maintained at pH greater than or equal to 8.0, this "prepore" dissociated to monomeric subunits upon treatment with SDS at room temperature, but treatment at pH less than or equal to 7 (or with beta-octylglucoside at pH 8.0) caused it to convert to an SDS-resistant pore-like form. Transition to this form involved major changes in the conformation of loop 2 of domain 2 (D2L2), as evidenced by (i) occlusion of a chymotrypsin site within D2L2 and (ii) excimer formation by pyrene groups linked to N306C within this loop. The pore-like form retained the capacity to bind anthrax toxin A moieties and cell surface receptors, but was unable to form pores in membranes or mediate translocation. Mutant PA(63) in which D2L2 had been deleted was inactive in pore formation and translocation but, like the prepore, was capable of forming heptamers that converted to an SDS-resistant form under acidic conditions. Our findings support a model of pore formation in which the D2L2 loops move to the membrane-proximal face of the heptamer and interact to form a 14-strand transmembrane beta-barrel. Concomitantly, domain 2 undergoes a major conformational rearrangement, independent of D2L2, that renders the heptamer resistant to dissociation by SDS. These results provide a basis for further exploration of the role of PA(63) in translocation of anthrax toxin's enzymic moieties across membranes.