7AD9

Structure of the Lifeact-F-actin complex

7AD9 の概要

• エントリーDOI: 10.2210/pdb7ad9/pdb
• EMDBエントリー: 11721
• 関連するBIRD辞書のPRD_ID: PRD_002307
• 分子名称: Lifeact, Actin, alpha skeletal muscle, Phalloidin, ... (6 entities in total)
• 機能のキーワード: cytoskeleton, actin, lifeact, labelling., structural protein
• 由来する生物種: Oryctolagus cuniculus (Rabbit); 詳細
• タンパク質・核酸の鎖数: 15
• 化学式量合計: 226962.96

構造登録者

Belyy, A.,Merino, F.,Sitsel, O.,Raunser, S. (登録日: 2020-09-14, 公開日: 2020-10-28, 最終更新日: 2020-12-02)

主引用文献

Belyy, A.,Merino, F.,Sitsel, O.,Raunser, S.
Structure of the Lifeact-F-actin complex.
Plos Biol., 18:e3000925-e3000925, 2020

PubMed Abstract: Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

PubMed: 33216759
DOI: 10.1371/journal.pbio.3000925

実験手法

ELECTRON MICROSCOPY (3.5 Å)