Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen
Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen
复制标题
DOI：
10.1074/jbc.m109.093195
复制
发表时间：
2010-03-12
影响因子：
4.8
通讯作者：
Collier, R. John
中科院分区：
文献类型：
--
作者：
Janowiak, Blythe E.;Fischer, Audrey;Collier, R. John
炭疽毒素的保护性抗原部分在内体膜中形成的多聚孔将毒素的酶部分易位至哺乳动物细胞的胞质区室。有证据表明，Phe(427) 残基的侧链在孔腔中彼此紧密靠近，并形成一种称为 Phe 钳的结构，催化易位过程。在本报告中，我们描述了用碱性或酸性氨基酸替换主要七聚孔的单个亚基中的 Phe427 的效果。在我们的标准测定中，在该位置掺入任何带电残基可抑制细胞毒性> = 1,000 倍，并对平面磷脂双层系统中的易位产生强烈抑制。 His 和 Glu 是最强的抑制残基，消除细胞毒性和易位。位置 427 处的碱性残基阻止 Phe 钳与易位底物相互作用以形成阻止离子通过的密封并加速底物从孔中的解离。相反，酸性残基允许形成密封，并且底物保持牢固结合，但可能通过与带正电的 N 末端片段的静电相互作用而阻止其通过。我们的研究结果与 Phe 钳在易位的布朗棘轮模型中的作用进行了讨论。
Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe427 in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >= 1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.