HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists
- The Scripps Research Institute, United States
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.
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Data fromfigshare, 10.6084/m9.figshare.8230685.
Article and author information
Author details
Timothy S Strutzenberg
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.
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.
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HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists
eLife 8:e47172.
https://doi.org/10.7554/eLife.47172
Further reading
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.
