Time-resolved structural analysis of tetanus toxin by Cryo-EM to elucidate the mechanism of membrane penetration

2024 Fiscal Year Final Research Report

• Project/Area Number: 23K23821
• Project/Area Number (Other): 22H02557 (2022-2023)
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Multi-year Fund (2024); Single-year Grants (2022-2023)
• Section: 一般
• Review Section: Basic Section 43020:Structural biochemistry-related
• Research Institution: Osaka University
• Principal Investigator: Inoue Tsuyoshi 大阪大学, 大学院薬学研究科, 教授 (20263204)
• Co-Investigator(Kenkyū-buntansha): 安居 輝人 北海道大学, 遺伝子病制御研究所, 博士研究員 (60283074)
• Project Period (FY): 2024-04-01 – 2025-03-31
• Keywords: 破傷風毒素 / クライオ電顕 / ダイナミクス / 抗体複合体 / 時分割解析

Outline of Final Research Achievements

Tetanus toxin (TeNT) is a potent neurotoxin that selectively targets the central nervous system, and elucidating its activation mechanism remains an important challenge. This study focused on the structural changes during membrane translocation, particularly the transition to a reduced state and the dissociation of disulfide bonds, and employed cryo-electron microscopy (cryo-EM) for analysis. Although the membrane-associated structures were diverse, the accumulation of sample preparation techniques and imaging conditions led to promising groundwork for future high-resolution studies. In parallel, the development of a visualization system using fluorescently labeled mutants and analysis of antibody-toxin complexes revealed part of the mechanism by which TeNT activation is suppressed. These findings are expected to contribute not only to the understanding of the CNS-specific activation mechanism of TeNT, but also to the development of effective neutralizing antibodies.

Free Research Field

構造生物学

Academic Significance and Societal Importance of the Research Achievements

破傷風毒素（TeNT）は中枢神経特異的に作用する強力な神経毒である。本研究では、S-S結合の乖離を伴う膜透過時の構造変化に注目し、クライオ電顕での解析を進めた。膜上構造の多様性に対応する技術的基盤を整備し、今後の高分解能解析に繋がる成果を得た。さらに、蛍光変異体による局在可視化系の構築や、抗体複合体解析により活性化阻害機構の一端を明らかにした。