7TDM

CryoEM Structure of sFab COP-2 Complex with human claudin-4 and Clostridium perfringens enterotoxin C-terminal domain

7TDM の概要

• エントリーDOI: 10.2210/pdb7tdm/pdb
• EMDBエントリー: 25834
• 分子名称: Claudin-4, Heat-labile enterotoxin B chain, COP-2 Fab Heavy chain, ... (4 entities in total)
• 機能のキーワード: claudin, cell adhesion, enterotoxin, fab, antibody fragment, membrane protein
• 由来する生物種: Homo sapiens (human); 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 85985.21

構造登録者

Vecchio, A.J. (登録日: 2022-01-01, 公開日: 2022-02-09, 最終更新日: 2024-11-06)

主引用文献

Orlando, B.J.,Dominik, P.K.,Roy, S.,Ogbu, C.P.,Erramilli, S.K.,Kossiakoff, A.A.,Vecchio, A.J.
Development, structure, and mechanism of synthetic antibodies that target claudin and Clostridium perfringens enterotoxin complexes.
J.Biol.Chem., 298:102357-102357, 2022

PubMed Abstract: Strains of Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflicts humans and domesticated animals, causing prevalent gastrointestinal illnesses. CpE's C-terminal domain (cCpE) binds cell surface receptors, followed by a restructuring of its N-terminal domain to form a membrane-penetrating β-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are known receptors for CpE and also control the architecture and function of cell-cell contacts (tight junctions) that create barriers to intercellular molecular transport. CpE binding and assembly disables claudin barrier function and induces cytotoxicity via β-pore formation, disrupting gut homeostasis; however, a structural basis of this process and strategies to inhibit the claudin-CpE interactions that trigger it are both lacking. Here, we used a synthetic antigen-binding fragment (sFab) library to discover two sFabs that bind claudin-4 and cCpE complexes. We established these sFabs' mode of molecular recognition and binding properties and determined structures of each sFab bound to claudin-4-cCpE complexes using cryo-EM. The structures reveal that the sFabs bind a shared epitope, but conform distinctly, which explains their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in binding changes, validating the structures, and uncovering the sFab's targeting mechanism. From these insights, we generated a model for CpE's claudin-bound β-pore that predicted sFabs would not prevent cytotoxicity, which we then verified in vivo. Taken together, this work demonstrates the development and mechanism of claudin/cCpE-binding sFabs that provide a framework and strategy for obstructing claudin/CpE assembly to treat CpE-linked gastrointestinal diseases.

PubMed: 35952760
DOI: 10.1016/j.jbc.2022.102357

実験手法

ELECTRON MICROSCOPY (6.9 Å)