Structure of the Lifeact-F-actin complex.
Actins
/ chemistry
Animals
Bacterial Toxins
/ chemistry
Binding Sites
Binding, Competitive
Cofilin 1
/ chemistry
Cryoelectron Microscopy
Fluorescent Dyes
/ chemistry
HEK293 Cells
Humans
Hydrophobic and Hydrophilic Interactions
In Vitro Techniques
Microfilament Proteins
/ chemistry
Microscopy, Confocal
Models, Molecular
Myosins
/ chemistry
Peptide Fragments
/ chemistry
Protein Engineering
Protein Interaction Domains and Motifs
Rabbits
Recombinant Fusion Proteins
/ chemistry
Saccharomyces cerevisiae Proteins
/ chemistry
Journal
PLoS biology
ISSN: 1545-7885
Titre abrégé: PLoS Biol
Pays: United States
ID NLM: 101183755
Informations de publication
Date de publication:
11 2020
11 2020
Historique:
received:
18
02
2020
accepted:
26
10
2020
revised:
04
12
2020
pubmed:
21
11
2020
medline:
5
1
2021
entrez:
20
11
2020
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 33216759
doi: 10.1371/journal.pbio.3000925
pii: PBIOLOGY-D-20-00395
pmc: PMC7717565
doi:
Substances chimiques
ABP140 protein, S cerevisiae
0
Actins
0
Bacterial Toxins
0
CFL1 protein, human
0
Cofilin 1
0
Fluorescent Dyes
0
Microfilament Proteins
0
Peptide Fragments
0
Recombinant Fusion Proteins
0
Saccharomyces cerevisiae Proteins
0
Myosins
EC 3.6.4.1
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e3000925Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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