5XWD

Crystal structure of the complex of 059-152-Fv and EGFR-ECD

5XWD の概要

• エントリーDOI: 10.2210/pdb5xwd/pdb
• 分子名称: Epidermal growth factor receptor, VH chain of 059-152, VL chain of 059-152, ... (6 entities in total)
• 機能のキーワード: antibody, receptor, complex, signaling protein
• 由来する生物種: Homo sapiens (Human); 詳細
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 102389.77

構造登録者

Matsuda, T.,Ito, T.,Shirouzu, M. (登録日: 2017-06-29, 公開日: 2018-02-28, 最終更新日: 2024-10-09)

主引用文献

Matsuda, T.,Ito, T.,Takemoto, C.,Katsura, K.,Ikeda, M.,Wakiyama, M.,Kukimoto-Niino, M.,Yokoyama, S.,Kurosawa, Y.,Shirouzu, M.
Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction
PLoS ONE, 13:e0193158-e0193158, 2018

PubMed Abstract: Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.

PubMed: 29462206
DOI: 10.1371/journal.pone.0193158

実験手法

X-RAY DIFFRACTION (2.894 Å)