Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single-cysteine variants labeled with the environmentally sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure.

破伤风神经毒素 (TeNT) 是一种 AB 毒素，具有三个功能域：内肽酶、易位 (HCT) 和受体结合。内体酸化触发 HCT 与膜相互作用并插入膜中，使内肽酶跨双层移位。尽管 HCT 的功能已明确定义，但其完成此任务的机制尚不清楚。为了深入了解局部和全局范围内的 HCT 膜相互作用，我们利用了一种分离的无带 HCT 变体 (bHCT)，它保留了从囊泡中释放钾离子的能力。为了检查哪些 bHCT 残基与膜相互作用，我们使用 47 个用环境敏感荧光团 NBD 标记的单半胱氨酸变体对 bHCT 表面进行了广泛采样。在中性 pH 值下，没有观察到任何变体的相互作用。相比之下，所有 NBD 标记的位置都报告了在酸性 pH 值和含有阴离子脂质的膜存在的情况下的环境变化。然后，我们使用圆二色性和内在荧光检查了插入的 bHCT 的构象。进入膜后，bHCT 主要保留α-螺旋二级结构，而三级结构表现出大量的重折叠。使用脂质附着猝灭剂表明，三个色氨酸残基中至少有一个深入渗透到膜的碳氢化合物核心，表明形成了 bHCT 跨膜构象。使用水病分析网络工具 MPEx 进一步探索了可能的构象拓扑，该工具识别出一个大的、潜在的α螺旋跨膜区域。 总而言之，光谱证据支持这样一个模型：酸化后，大多数 TeNT bHCT 进入膜，同时三级结构发生变化。