Schiffmiller Aviva, Finkelstein Alan
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
J Mol Biol. 2015 Mar 27;427(6 Pt A):1211-23. doi: 10.1016/j.jmb.2014.06.016. Epub 2014 Jul 1.
The tripartite anthrax toxin consists of protective antigen, lethal factor (LF), and edema factor. PA63 (the 63-kDa, C-terminal part of protective antigen) forms heptameric channels in cell membranes that allow for the transport of LF and edema factor into the cytosol. These channels are mushroom shaped, with a ring of seven phenylalanine residues (known as the phenylalanine clamp) lining the junction between the cap and the stem. It is known that when LF is translocated through the channel, the phenylalanine clamp creates a seal that causes an essentially complete block of conduction. In order to examine ion conductance in the stem of the channel, we used Venus yellow fluorescent protein as a molecular stopper to trap LFN (the 30-kDa, 263-residue N-terminal segment of LF), as well as various truncated constructs of LFN, in mutant channels in which the phenylalanine clamp residues were mutated to alanines. Here we present evidence that ion movement occurs within the channel stem (but is stopped, of course, at the phenylalanine clamp) during protein translocation. Furthermore, we also propose that the lower region of the stem plays an important role in securing peptide chains during translocation.
三联体炭疽毒素由保护性抗原、致死因子(LF)和水肿因子组成。PA63(保护性抗原的63 kDa C末端部分)在细胞膜中形成七聚体通道,使LF和水肿因子能够转运到细胞质中。这些通道呈蘑菇状,在帽部和柄部的连接处有一圈七个苯丙氨酸残基(称为苯丙氨酸钳)。已知当LF通过通道转运时,苯丙氨酸钳会形成一个密封,导致传导基本完全阻断。为了检测通道柄部的离子传导,我们使用金星黄色荧光蛋白作为分子塞子,将LFN(LF的30 kDa、263个残基的N末端片段)以及LFN的各种截短构建体捕获在苯丙氨酸钳残基突变为丙氨酸的突变通道中。在此我们提供证据表明,在蛋白质转运过程中,离子在通道柄部内移动(但当然在苯丙氨酸钳处停止)。此外,我们还提出,柄部的下部区域在转运过程中固定肽链方面起着重要作用。