Structural basis for the Rad6 activation by the Bre1 N-terminal domain

  1. Meng Shi
  2. Jiaqi Zhao
  3. Simin Zhang
  4. Wei Huang
  5. Mengfei Li
  6. Xue Bai
  7. Wenxue Zhang
  8. Kai Zhang
  9. Xuefeng Chen  Is a corresponding author
  10. Song Xiang  Is a corresponding author
  1. Tianjin Medical University, China
  2. Wuhan University, China
  3. Tianjin Medical University General Hospital, China

Abstract

The mono-ubiquitination of the histone protein H2B (H2Bub1) is a highly conserved histone post-translational modification that plays critical roles in many fundamental processes. In yeast, this modification is catalyzed by the conserved Bre1-Rad6 complex. Bre1 contains a unique N-terminal Rad6 binding domain (RBD), how it interacts with Rad6 and contributes to the H2Bub1 catalysis is unclear. Here, we present crystal structure of the Bre1 RBD-Rad6 complex and structure-guided functional studies. Our structure provides a detailed picture of the interaction between the dimeric Bre1 RBD and a single Rad6 molecule. We further found that the interaction stimulates Rad6's enzymatic activity by allosterically increasing its active site accessibility and likely contribute to the H2Bub1 catalysis through additional mechanisms. In line with these important functions, we found that the interaction is crucial for multiple H2Bub1-regulated processes. Our study provides molecular insights into the H2Bub1 catalysis.

Data availability

Diffraction data and refined structures of crystal forms 1 and 2 of the KlBre1 RBD-Rad6 complex have been deposited into the protein data bank (www.rcsb.org), with accession codes 7W75 and 7W76, respectively. All data generated or analysed during this study are included in the manuscript and supporting file; source data files are provided for Figures 1-5, figure 1 figure supplement 3 and figure 3 figure supplement 1.

The following data sets were generated

Article and author information

Author details

  1. Meng Shi

    Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China
    Competing interests
    The authors declare that no competing interests exist.
  2. Jiaqi Zhao

    Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China
    Competing interests
    The authors declare that no competing interests exist.
  3. Simin Zhang

    College of Life Sciences, Wuhan University, Wuhan, China
    Competing interests
    The authors declare that no competing interests exist.
  4. Wei Huang

    Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China
    Competing interests
    The authors declare that no competing interests exist.
  5. Mengfei Li

    College of Life Sciences, Wuhan University, Wuhan, China
    Competing interests
    The authors declare that no competing interests exist.
  6. Xue Bai

    Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China
    Competing interests
    The authors declare that no competing interests exist.
  7. Wenxue Zhang

    Department of Radiation Oncology, Tianjin Medical University General Hospital, Tianjin, China
    Competing interests
    The authors declare that no competing interests exist.
  8. Kai Zhang

    Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China
    Competing interests
    The authors declare that no competing interests exist.
  9. Xuefeng Chen

    College of Life Sciences, Wuhan University, Wuhan, China
    For correspondence
    xfchen@whu.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
  10. Song Xiang

    Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China
    For correspondence
    xiangsong@tmu.edu.cn
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9314-4684

Funding

National Natural Science Foundation of China (32271259,32071205 and 31870769)

  • Song Xiang

National Natural Science Foundation of China (32070573 and 31872808)

  • Xuefeng Chen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Xiaobing Shi, Van Andel Institute, United States

Version history

  1. Received: October 12, 2022
  2. Preprint posted: October 24, 2022 (view preprint)
  3. Accepted: March 10, 2023
  4. Accepted Manuscript published: March 13, 2023 (version 1)
  5. Version of Record published: March 23, 2023 (version 2)

Copyright

© 2023, Shi 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

  • 630
    Page views
  • 147
    Downloads
  • 2
    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. Meng Shi
  2. Jiaqi Zhao
  3. Simin Zhang
  4. Wei Huang
  5. Mengfei Li
  6. Xue Bai
  7. Wenxue Zhang
  8. Kai Zhang
  9. Xuefeng Chen
  10. Song Xiang
(2023)
Structural basis for the Rad6 activation by the Bre1 N-terminal domain
eLife 12:e84157.
https://doi.org/10.7554/eLife.84157

Further reading

    1. Structural Biology and Molecular Biophysics
    Xiaoxuan Lin, Patrick R Haller ... Tobin R Sosnick
    Research Article

    Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin’s voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen–deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl binding. This event shortens the TM3–TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin’s mechanical expansion. We observe helix fraying at prestin’s anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin’s fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin’s unique voltage-sensing mechanism and electromotility.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Daniel Muñoz-Reyes, Levi J McClelland ... Maria Jose Sanchez-Barrena
    Research Article

    The Neuronal Calcium Sensor 1, an EF-hand Ca2+ binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Ga have revealed how Ric-8A phosphorylation promotes Ga recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Ga subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+ signals and phosphorylation events that promote the interaction of Ric-8A with Ga. Our data show that the binding of NCS-1 and Ga to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Ga when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+ concentration. Finally, the high-resolution crystallographic data reported here define the NCS-1/Ric-8A interface and will allow the development of therapeutic synapse function regulators with improved activity and selectivity.