Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan.
Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan.
Sci Adv. 2019 Jul 3;5(7):eaax0495. doi: 10.1126/sciadv.aax0495. eCollection 2019 Jul.
Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K channels. The docking model has been elucidated, but it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.
蝎毒素中的 Agitoxin-2（AgTx2）是一种有效的 K 通道阻断剂。已经阐明了其对接模型，但目前尚不清楚结合动力学是否由二态模型（AgTx2 结合态和 AgTx2 非结合态）或更复杂的机制（如诱导契合或构象选择）来描述。在这里，我们使用高速原子力显微镜观察了 AgTx2 与 KcsA 通道的结合动力学。从 AgTx2 与通道重复结合和解离的图像中，通过单分子动力学分析揭示了通道对 AgTx2 的亲和力在持续结合过程中增加，而在持续解离过程中降低。我们提出了一个四态模型，包括通道的高亲和态和低亲和态，以及相关的速率常数。诱导契合途径占主导地位，并将结合速度加快了 400 倍。这是首次实时分析蝎毒素结合的解析成像，适用于包括通道配体、DNA 修饰蛋白和抗原抗体复合物在内的各种生物动力学。
