1. Structural Biology and Molecular Biophysics
Download icon

Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

  1. Dan Tan
  2. Qiang Li
  3. Mei-Jun Zhang
  4. Chao Liu
  5. Chengying Ma
  6. Pan Zhang
  7. Yue-He Ding
  8. Sheng-Bo Fan
  9. Li Tao
  10. Bing Yang
  11. Xiangke Li
  12. Shoucai Ma
  13. Junjie Liu
  14. Boya Feng
  15. Xiaohui Liu
  16. Hong-Wei Wang
  17. Si-Min He
  18. Ning Gao
  19. Keqiong Ye
  20. Meng-Qiu Dong  Is a corresponding author
  21. Xiaoguang Lei
  1. Peking Union Medical College, Chinese Academy of Medical Sciences, China
  2. National Institute of Biological Sciences, China
  3. Institute of Computing Technology, Chinese Academy of Sciences, China
  4. Tsinghua University, China
  5. Chinese Academy of Medical Sciences, Peking Union Medical College, China
  6. Tianjin University, China
Tools and Resources
  • Cited 48
  • Views 7,142
  • Annotations
Cite this article as: eLife 2016;5:e12509 doi: 10.7554/eLife.12509

Abstract

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.

Article and author information

Author details

  1. Dan Tan

    Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  2. Qiang Li

    National Institute of Biological Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  3. Mei-Jun Zhang

    National Institute of Biological Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Chao Liu

    Key Lab of Intelligent Information Processing of Chinese Academy of Sciences, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  5. Chengying Ma

    Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  6. Pan Zhang

    Graduate Program, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  7. Yue-He Ding

    Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  8. Sheng-Bo Fan

    Key Lab of Intelligent Information Processing of Chinese Academy of Sciences, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  9. Li Tao

    Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  10. Bing Yang

    National Institute of Biological Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  11. Xiangke Li

    National Institute of Biological Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  12. Shoucai Ma

    National Institute of Biological Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  13. Junjie Liu

    Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  14. Boya Feng

    Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  15. Xiaohui Liu

    College of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China
    Competing interests
    The authors declare that no competing interests exist.
  16. Hong-Wei Wang

    Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  17. Si-Min He

    Key Lab of Intelligent Information Processing of Chinese Academy of Sciences, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  18. Ning Gao

    Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  19. Keqiong Ye

    National Institute of Biological Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  20. Meng-Qiu Dong

    Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China
    For correspondence
    dongmengqiu@nibs.ac.cn
    Competing interests
    The authors declare that no competing interests exist.
  21. Xiaoguang Lei

    National Institute of Biological Sciences, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Brian Chait

Publication history

  1. Received: October 23, 2015
  2. Accepted: February 26, 2016
  3. Accepted Manuscript published: March 8, 2016 (version 1)
  4. Version of Record published: March 18, 2016 (version 2)
  5. Version of Record updated: September 20, 2016 (version 3)

Copyright

© 2016, Tan 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

  • 7,142
    Page views
  • 2,047
    Downloads
  • 48
    Citations

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

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)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Structural Biology and Molecular Biophysics
    Lily Goodyer Sait et al.
    Research Article

    Voltage-gated sodium channels are targets for a range of pharmaceutical drugs developed for treatment of neurological diseases. Cannabidiol (CBD), the non-psychoactive compound isolated from cannabis plants, was recently approved for treatment of two types of epilepsy associated with sodium channel mutations. This study used high resolution X-ray crystallography to demonstrate the detailed nature of the interactions between CBD and the NavMs voltage-gated sodium channel, and electrophysiology to show the functional effects of binding CBD to these channels. CBD binds at a novel site at the interface of the fenestrations and the central hydrophobic cavity of the channel. Binding at this site blocks the transmembrane-spanning sodium ion translocation pathway, providing a molecular mechanism for channel inhibition. Modelling studies suggest why the closely-related psychoactive compound tetrahydrocannabinol may not have the same effects on these channels. Finally, comparisons are made with the TRPV2 channel, also recently proposed as a target site for CBD. In summary, this study provides novel insight into a possible mechanism for CBD interactions with sodium channels.

    1. Structural Biology and Molecular Biophysics
    Xue Fei et al.
    Research Article

    When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In E. coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer-degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.