AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions
- Ehime University, Japan
- University of Shizuoka, Japan
- Tokushima University, Japan
Abstract
Proximity biotinylation based on Escherichia coli BirA enzymes like BioID (BirA*) and TurboID is a key technology for identifying proteins interacting with a target protein in a cell or organism. However, there have been some improvements in the enzymes for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins like AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-IκBα biotinylated the endogenous CUL4 and RBX1 in the CRL4CRBN complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analysing protein–protein interactions.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3, 4, and 6.
Article and author information
Author details
Funding
Japan Agency for Medical Research and Development (JP19am0101077)
- Tatsuya Sawasaki
Japan Society for the Promotion of Science (JP16H06579)
- Tatsuya Sawasaki
Japan Society for the Promotion of Science (JP16H04729)
- Tatsuya Sawasaki
Japan Society for the Promotion of Science (JP19H03218)
- Tatsuya Sawasaki
Japan Society for the Promotion of Science (18KK0229)
- Hidetaka Kosako
Japan Society for the Promotion of Science (19H04966)
- Hidetaka Kosako
Takeda Science Foundation
- Tatsuya Sawasaki
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Kido et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 25,236
- views
-
- 3,487
- downloads
-
- 97
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions
eLife 9:e54983.
https://doi.org/10.7554/eLife.54983
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
In the bacterium M. smegmatis, an enzyme called MftG allows the cofactor mycofactocin to transfer electrons released during ethanol metabolism to the electron transport chain.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Liquid-liquid phase separation (LLPS) involving intrinsically disordered protein regions (IDRs) is a major physical mechanism for biological membraneless compartmentalization. The multifaceted electrostatic effects in these biomolecular condensates are exemplified here by experimental and theoretical investigations of the different salt- and ATP-dependent LLPSs of an IDR of messenger RNA-regulating protein Caprin1 and its phosphorylated variant pY-Caprin1, exhibiting, for example, reentrant behaviors in some instances but not others. Experimental data are rationalized by physical modeling using analytical theory, molecular dynamics, and polymer field-theoretic simulations, indicating that interchain ion bridges enhance LLPS of polyelectrolytes such as Caprin1 and the high valency of ATP-magnesium is a significant factor for its colocalization with the condensed phases, as similar trends are observed for other IDRs. The electrostatic nature of these features complements ATP’s involvement in π-related interactions and as an amphiphilic hydrotrope, underscoring a general role of biomolecular condensates in modulating ion concentrations and its functional ramifications.
