Reply to Yamini and Nestorovich: Alternate clamped states of the anthrax toxin protective antigen channel.
Reply to Yamini and Nestorovich: Alternate clamped states of the anthrax toxin protective antigen channel.
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回复 Yamini 和 Nestorovich：炭疽毒素保护性抗原通道的交替钳位状态。
DOI：
10.1073/pnas.1702212114
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发表时间：
2017
影响因子：
11.1
通讯作者：
Krantz,BryanA
中科院分区：
文献类型：
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作者：
Krantz,BryanA
他们提出的第一个问题是电导亚状态是否是PA通道苯丙氨酸钳（Phenylalanine clamp）位点的钳位状态。我们认为是这种情况，因为当β-钳从苯丙氨酸突变为丙氨酸时，通道的两个亚基之间的电流差从野生型的0.7pA增加到PA F427 A突变体的2.1pA。因为钳位是沟道中的主要电导瓶颈，所以该结构的膨胀可以解释电流差异。由于钳位对肽移位机制至关重要，我们将这两种状态命名为未钳位空扩张和钳位空，其中空表示通道不含肽。提出的第二点与PA通道的快速闪烁关闭有关。我们没有直接研究这个过程，但是，我们归因于一些较慢的时间尺度关闭的PA通道的缓冲液或双层设置中的污染物。这种归因也应该包括通道本身的虚假门控过程，我们感谢亚米尼和涅斯托罗维奇（1）英勇地表明这些关闭发生在高度纯净的水中。尽管如此，这一点相对较小，与论文的中心论点无关。Yamini和Nestorovich（1）还提出，在不存在肽的情况下，两种电导亚态的时间尺度是相关的。他们引用了一项研究，其中较低的电导状态仅“偶尔”被填充。我们认为，这个时间尺度与含有肽的系统无关，因为肽变构改变了系统在松弛和绷紧状态下的平衡解离常数> 300倍。一个相对罕见的外观的亚电导状态是符合结合解离常数的这种大的变化。他们还指出，β-环糊精与系统的两个亚电导态的结合在其平衡解离常数方面是不可区分的。对于β-环糊精可能是正确的，但对于肽不一定是这样。我们认为，对于肽测量的变构效应对于可以采用α-螺旋的肽是独特的，其中β-环糊精的形状与肽α-螺旋有很大不同。
The first point they raised was whether the conductance substate was a clamped state of the PA channel’s phenylalanine clamp (ϕ-clamp) site. We believe that such is the case because when the ϕ-clamp was mutated from phenylalanine to alanine, the current difference between the two substates of the channel increased from 0.7 pA for wild type to 2.1 pA for the PA F427A mutant. Because the ϕ-clamp is the major conductance bottleneck in the channel, dilation of this structure may explain the current difference. Because the ϕ-clamp is critical to the mechanism of peptide translocation, we named the two states unclamped empty dilated and clamped empty, where empty denotes that the channel is peptide-free. The second point raised was related to a fastflickering closure of the PA channel. We did not study this process directly; however, we attributed some of the slower time-scale closures of the PA channel to contaminants in the buffer or bilayer setup. This attribution should have included a spurious gating process in the channel itself as well, and we thank Yamini and Nestorovich (1) for heroically showing that these closures occur in highly purified water. Nonetheless, this point is relatively minor and has no bearing on the central arguments of the paper.Yamini and Nestorovich (1) also suggest the time scales of the two conductance substates were relevant in the absence of peptide. They cited a study where the lower conductance state was populated only “on occasion.” We contend that this time scale is not relevant to the system containing the peptide, because the peptide allosterically alters the system> 300-fold in terms of the equilibrium dissociation constants for the relaxed and taut states of the system. A relatively rare appearance of the subconductance state is in line with this large change in binding dissociation constants. They additionally cited that the binding of β-cyclodextrins to the two subconductance states of the system was indistinguishable in terms of their equilibrium dissociation constants. What may be true for β-cyclodextrins is not necessarily the case for peptides. We contend that the allosteric effect measured for peptides is unique for peptides that can adopt an α-helix, where the shape of a β-cyclodextrin is much different from a peptide α-helix.