Zhang Chun-Ming, Imoto Yoshihiro, Hikima Takaaki, Inoue Tsuyoshi
Graduate School of Pharmaceutical Science, Osaka University, Suita, 565-0871 Osaka, Japan.
Advanced Photon Technology Division, RIKEN SPring-8 Center, Sayo-gun, 679-6148, Japan.
J Struct Biol X. 2021 Jan 30;5:100045. doi: 10.1016/j.yjsbx.2021.100045. eCollection 2021.
Although the tetanus neurotoxin (TeNT) delivers its protease domain (LC) across the synaptic vesicle lumen into the cytosol via its receptor binding domain (H) and translocation domain (H), the molecular mechanism coordinating this membrane translocation remains unresolved. Here, we report the high-resolution crystal structures of full-length reduced TeNT (rTeNT, 2.3 Å), TeNT isolated H (TeNT/iH, 2.3 Å), TeNT isolated H (TeNT/iH, 1.5 Å), together with the solution structures of TeNT/iH and beltless TeNT/iH (TeNT/blH). TeNT undergoes significant domains rotation of the H and LC were demonstrated by structural comparison of rTeNT and non-reduced-TeNT (nrTeNT). A linker loop connects the H and H is essential for the self-domain rotation of TeNT. The TeNT-specific C869-C1093 disulfide bond is sensitive to the redox environment and its disruption provides linker loop flexibility, which enables domain arrangement of rTeNT distinct from that of nrTeNT. Furthermore, the mobility of C869 in the linker loop and the sensitivity to redox condition of C1093 were confirmed by crystal structure analysis of TeNT/iH. On the other hand, the structural flexibility of H was investigated by crystallographic and solution scattering analyses. It was found that the region (residues 698-769), which follows the translocation region had remarkable change in TeNT/iH. Besides, the so-called belt region has a high propensity to swing around the upper half of TeNT/iH at acidic pH. It provides the first overview of the dynamics of the Belt in solution. These newly obtained structural information that shed light on the transmembrane mechanism of TeNT.
尽管破伤风神经毒素（TeNT）通过其受体结合结构域（H）和转运结构域（H）将其蛋白酶结构域（轻链，LC）穿过突触小泡腔递送至细胞质中，但协调这种膜转运的分子机制仍未得到解决。在此，我们报告了全长还原型TeNT（rTeNT，2.3 Å）、分离的TeNT H结构域（TeNT/iH，2.3 Å）、TeNT/iH（1.5 Å）的高分辨率晶体结构，以及TeNT/iH和无带TeNT/iH（TeNT/blH）的溶液结构。通过rTeNT和非还原型TeNT（nrTeNT）的结构比较表明，TeNT的H结构域和LC结构域发生了显著的旋转。连接H和H'的连接环对于TeNT的自身结构域旋转至关重要。TeNT特有的C869-C1093二硫键对氧化还原环境敏感，其断裂提供了连接环的灵活性，这使得rTeNT的结构域排列与nrTeNT不同。此外，通过TeNT/iH的晶体结构分析证实了连接环中C869的流动性以及C1093对氧化还原条件的敏感性。另一方面，通过晶体学和溶液散射分析研究了H'的结构灵活性。发现在TeNT/iH中，转运区域之后的区域（残基698-769）有显著变化。此外，所谓的带区在酸性pH下有很高的倾向围绕TeNT/iH的上半部分摆动。它首次概述了溶液中带区的动力学。这些新获得的结构信息为TeNT的跨膜机制提供了线索。
