Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins
Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins
复制标题
DOI：
10.1085/jgp.201010578
复制
发表时间：
2011-04-01
影响因子：
3.8
通讯作者：
Finkelstein, Alan
中科院分区：
文献类型：
--
作者：
Basilio, Daniel;Jennings-Antipov, Laura D.;Finkelstein, Alan
炭疽毒素由三种蛋白质组成：致死因子（LF）、水肿因子（EF）和保护性抗原（PA）。后者在宿主细胞的内体膜上形成一个七聚体通道（PA(63)）(7)，使得前两者（它们是酶）能够转移到细胞质中。结合到平面双层膜中的（PA(63)）(7)形成一个通道，该通道可转移LF和EF，且N末端在前。该通道呈蘑菇状，其帽部含有EF和LF的结合位点，还有一个约100埃长、15埃宽的茎部。为了使蛋白质能够通过茎部，它们显然必须去折叠，但是二级结构是否保留呢？为了回答这个问题，我们开发了一种在通道内捕获正在转移的蛋白质的多肽链的方法，并确定了能够穿过通道的最少残基数。我们在LF(N)（LF的263个残基的N末端部分）的N末端连接了一个生物素，并在其他位置连接了一个分子塞。如果从N末端到塞子的距离足够长以穿过通道，添加到另一侧的链霉亲和素就会结合N末端的生物素，从而将蛋白质捕获在通道内；如果这个距离不够长，链霉亲和素就不会结合N末端的生物素，蛋白质也就不会被捕获。捕获率取决于驱动力（电压）、施加驱动力的时间长度以及N末端和塞子之间的残基数。通过改变塞子的位置，我们确定了跨越通道所需的最少残基数。我们得出结论，LF(N)在转移时采用伸展链构象；即通道使蛋白质的二级结构去折叠。我们还表明，通道不仅能够按正常方向转移LF(N)，而且至少能够部分地按相反方向转移LF(N)。
Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA(63))(7), in the host cell's endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA(63))(7) incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an similar to 100 angstrom-long, 15 angstrom-wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LF(N) (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LF(N) adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LF(N) in the normal direction but also can, at least partially, translocate LF(N) in the opposite direction.