1. Biochemistry and Chemical Biology
Download icon

HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

Research Article
  • Cited 6
  • Views 1,607
  • Annotations
Cite this article as: eLife 2019;8:e47172 doi: 10.7554/eLife.47172

Abstract

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism to inflammation and cancer. Synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology. Nearly half of NRs do not have specific cognate ligands (termed orphan NRs) and it's unclear if they possess ligand dependent activities. Here we demonstrate that ligand-dependent action of the orphan RORγ can be defined by selectively disrupting putative endogenous-but not synthetic-ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings, corroborated with X-ray co-crystallography and site-directed mutagenesis, collectively reveal the structural determinants of RORγ activation, which is critical for designing RORγ agonists for cancer immunotherapy.

Data availability

Due to their large size, all the raw data from MS analysis is available from the authors. HDX Workbench outputs files have been uploaded to figshare (10.6084/m9.figshare.8230685) along with a treated data summary which summarizes the findings from the raw data.

The following data sets were generated

Article and author information

Author details

  1. Timothy S Strutzenberg

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0598-534X
  2. Ruben D Garcia-Ordonez

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Scott J Novick

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. HaJeung Park

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Mi Ra Chang

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Christelle Doebellin

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Yuanjun He

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Rémi Patouret

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Theodore M Kamenecka

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Patrick R Griffin

    Department of Molecular Medicine, The Scripps Research Institute, Jupiter, United States
    For correspondence
    pgriffin@scripps.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3404-690X

Funding

National Cancer Institute

  • Patrick R Griffin

National Institute of General Medical Sciences

  • Timothy S Strutzenberg

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

Reviewing Editor

  1. Peter Tontonoz, University of California, Los Angeles, United States

Publication history

  1. Received: March 27, 2019
  2. Accepted: June 1, 2019
  3. Accepted Manuscript published: June 7, 2019 (version 1)
  4. Version of Record published: June 17, 2019 (version 2)
  5. Version of Record updated: October 18, 2019 (version 3)

Copyright

© 2019, Strutzenberg 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

  • 1,607
    Page views
  • 234
    Downloads
  • 6
    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)

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. Biochemistry and Chemical Biology
    Xavier Portillo et al.
    Research Article Updated

    An RNA polymerase ribozyme that has been the subject of extensive directed evolution efforts has attained the ability to synthesize complex functional RNAs, including a full-length copy of its own evolutionary ancestor. During the course of evolution, the catalytic core of the ribozyme has undergone a major structural rearrangement, resulting in a novel tertiary structural element that lies in close proximity to the active site. Through a combination of site-directed mutagenesis, structural probing, and deep sequencing analysis, the trajectory of evolution was seen to involve the progressive stabilization of the new structure, which provides the basis for improved catalytic activity of the ribozyme. Multiple paths to the new structure were explored by the evolving population, converging upon a common solution. Tertiary structural remodeling of RNA is known to occur in nature, as evidenced by the phylogenetic analysis of extant organisms, but this type of structural innovation had not previously been observed in an experimental setting. Despite prior speculation that the catalytic core of the ribozyme had become trapped in a narrow local fitness optimum, the evolving population has broken through to a new fitness locale, raising the possibility that further improvement of polymerase activity may be achievable.