Electrostatic ratchet in the protective antigen channel promotes anthrax toxin translocation.
Electrostatic ratchet in the protective antigen channel promotes anthrax toxin translocation.
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DOI：
10.1074/jbc.m112.419598
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发表时间：
2012-12-21
期刊：
影响因子：
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通讯作者：
Krantz BA
中科院分区：
文献类型：
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作者：
Wynia-Smith SL;Brown MJ;Chirichella G;Kemalyan G;Krantz BA
背景：棘轮是展开和运输生物聚合物的分子机器的关键特征。结果：通过实验鉴定了炭疽毒素蛋白转位酶中的静电棘轮并建立了模型。结论：炭疽毒素转座酶以静电棘轮利用质子动力。意义：本报告描述了一种静电棘轮元件，对质子动力驱动的易位至关重要。蛋白质易位的动力冲程和布朗棘轮机制的核心是分子马达纠正或棘轮非平衡波动以沿特定轴运输底物蛋白质的过程。我们以炭疽毒素为模型研究棘轮机制。炭疽毒素是一种由保护性抗原（PA）组分组成的三方毒素，它是一种跨膜转位酶，它在质子动力（PMF）作用下将另外两种酶组分致死因子（LF）和水肿因子（EF）易位到宿主细胞的细胞质中。LF和EF的pa结合域（LFN和EFN）具有相同的折叠和相似的溶液稳定性；然而，EFN的转运速度比LFN慢10 - 200倍，这取决于PMF的电势（Δψ）和化学势（ΔpH）组成。通过对LFN/EFN嵌合体蛋白的分析，我们确定了两个由带电序列组成的10个残基盒，它们是导致EFN易位动力学受损的原因。这些磁带具有非特异性静电要求：令人惊讶的是，当由Δψ或ΔpH驱动时，它们更喜欢酸性残留物；第二种只有在Δψ驱动下才需要基本残留物。通过建模和实验，我们确定了PA通道中负责电荷选择性的带电表面。带电表面锁住衬底并促进pmf驱动的传输。我们在通道中提出了一个静电棘轮，由带电残基的相反环组成，通过与衬底中的带电盒相互作用来强制方向性，从而产生足以驱动展开的力。
Background: Ratchets are key features in molecular machines that unfold and transport biopolymers. Results: An electrostatic ratchet in the anthrax toxin protein translocase was experimentally identified and modeled. Conclusion: The anthrax toxin translocase harnesses the proton motive force with an electrostatic ratchet. Significance: This report describes an electrostatic ratchet element critical to proton motive force-driven translocation. Central to the power-stroke and Brownian-ratchet mechanisms of protein translocation is the process through which nonequilibrium fluctuations are rectified or ratcheted by the molecular motor to transport substrate proteins along a specific axis. We investigated the ratchet mechanism using anthrax toxin as a model. Anthrax toxin is a tripartite toxin comprised of the protective antigen (PA) component, a homooligomeric transmembrane translocase, which translocates two other enzyme components, lethal factor (LF) and edema factor (EF), into the cytosol of the host cell under the proton motive force (PMF). The PA-binding domains of LF and EF (LFN and EFN) possess identical folds and similar solution stabilities; however, EFN translocates ∼10–200-fold slower than LFN, depending on the electrical potential (Δψ) and chemical potential (ΔpH) compositions of the PMF. From an analysis of LFN/EFN chimera proteins, we identified two 10-residue cassettes comprised of charged sequence that were responsible for the impaired translocation kinetics of EFN. These cassettes have nonspecific electrostatic requirements: one surprisingly prefers acidic residues when driven by either a Δψ or a ΔpH; the second requires basic residues only when driven by a Δψ. Through modeling and experiment, we identified a charged surface in the PA channel responsible for charge selectivity. The charged surface latches the substrate and promotes PMF-driven transport. We propose an electrostatic ratchet in the channel, comprised of opposing rings of charged residues, enforces directionality by interacting with charged cassettes in the substrate, thereby generating forces sufficient to drive unfolding.