AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions

  1. Kohki Kido
  2. Satoshi Yamanaka
  3. Shogo Nakano
  4. Kou Motani
  5. Souta Shinohara
  6. Akira Nozawa
  7. Hidetaka Kosako
  8. Sohei Ito
  9. Tatsuya Sawasaki  Is a corresponding author
  1. Ehime University, Japan
  2. University of Shizuoka, Japan
  3. 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

  1. Kohki Kido

    Proteo-Science Center, Ehime University, Matsuyama, Japan
    Competing interests
    The authors declare that no competing interests exist.
  2. Satoshi Yamanaka

    Proteo-Science Center, Ehime University, Matsuyama, Japan
    Competing interests
    The authors declare that no competing interests exist.
  3. Shogo Nakano

    Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan
    Competing interests
    The authors declare that no competing interests exist.
  4. Kou Motani

    Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan
    Competing interests
    The authors declare that no competing interests exist.
  5. Souta Shinohara

    Proteo-Science Center, Ehime University, Matsuyama, Japan
    Competing interests
    The authors declare that no competing interests exist.
  6. Akira Nozawa

    Proteo-Science Center, Ehime University, Matsuyama, Japan
    Competing interests
    The authors declare that no competing interests exist.
  7. Hidetaka Kosako

    Fujii Memorial Institute of Medical Sciences, Tokushima University, Tokushima, Japan
    Competing interests
    The authors declare that no competing interests exist.
  8. Sohei Ito

    Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan
    Competing interests
    The authors declare that no competing interests exist.
  9. Tatsuya Sawasaki

    Proteo-Science Center, Ehime University, Matsuyama, Japan
    For correspondence
    sawasaki@ehime-u.ac.jp
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7952-0556

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.

Reviewing Editor

  1. Volker Dötsch, Goethe University, Germany

Publication history

  1. Received: January 8, 2020
  2. Accepted: May 7, 2020
  3. Accepted Manuscript published: May 11, 2020 (version 1)
  4. Version of Record published: June 18, 2020 (version 2)

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

  • 13,590
    Page views
  • 1,954
    Downloads
  • 23
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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)

  1. Kohki Kido
  2. Satoshi Yamanaka
  3. Shogo Nakano
  4. Kou Motani
  5. Souta Shinohara
  6. Akira Nozawa
  7. Hidetaka Kosako
  8. Sohei Ito
  9. Tatsuya Sawasaki
(2020)
AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions
eLife 9:e54983.
https://doi.org/10.7554/eLife.54983

Further reading

    1. Biochemistry and Chemical Biology
    Tiantian Wei et al.
    Research Article Updated

    The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process. These studies collectively further expand our understanding of DYRK2 and provide a valuable tool to pinpoint its biological function.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Lukas P Feilen et al.
    Research Article

    Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme´s catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.