Identification of a pre-active conformation of a pentameric channel receptor
- Institut Pasteur, France
- Weill Cornell Medicine, United States
- Université Paris-Sud, France
- Inserm U1024, CNRS 8197, Institute of Biology, Ecole Normale Supérieure, France
Abstract
Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical signalling through global allosteric transitions. Despite the existence of several high-resolution structures of pLGICs, their dynamical properties remain elusive. Using the proton-gated channel GLIC, we engineered multiple fluorescent reporters, each incorporating a bimane and a tryptophan/tyrosine, whose close distance causes fluorescence quenching. We show that proton application causes a global compaction of the extracellular subunit interface, coupled to an outward motion of the M2-M3 loop near the channel gate. These movements are highly similar in lipid vesicles and detergent micelles. These reorganizations are essentially completed within 2 ms and occur without channel opening at low proton concentration, indicating that they report a pre-active intermediate state in the transition pathway towards activation. This provides a template to investigate the gating of eukaryotic neurotransmitter receptors, for which intermediate states also participate in activation.
Article and author information
Author details
Pierre-Jean Corringer
Funding
Agence Nationale de la Recherche (Pentagate)
- Anaïs Menny
- Solène N Lefebvre
- Emmanuelle Drège
- Zaineb Fourati
- Marc Delarue
- Delphine Joseph
- Pierre-Jean Corringer
Centre National de la Recherche Scientifique (UMR 3571)
- Anaïs Menny
- Solène N Lefebvre
- Delphine Joseph
Institut Pasteur
- Anaïs Menny
- Solène N Lefebvre
- Zaineb Fourati
- Marc Delarue
- Pierre-Jean Corringer
Université Pierre et Marie Curie (PhD student fellowship)
- Anaïs Menny
- Solène N Lefebvre
Fondation pour la Recherche Médicale (DEQ20140329497)
- Anaïs Menny
- Pierre-Jean Corringer
National Institutes of Health (R01 GM088352)
- Crina M Nimigean
Centre National de la Recherche Scientifique (UMR 8076)
- Emmanuelle Drège
- Delphine Joseph
Centre National de la Recherche Scientifique (UMR 3528)
- Zaineb Fourati
- Marc Delarue
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Menny 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
-
- 2,040
- views
-
- 524
- downloads
-
- 43
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
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)
Identification of a pre-active conformation of a pentameric channel receptor
eLife 6:e23955.
https://doi.org/10.7554/eLife.23955
Further reading
-
- Structural Biology and Molecular Biophysics
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous and synthetic ligands, including covalent antagonist inhibitors GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent inhibitors weaken—but do not prevent—the binding of other ligands via an allosteric mechanism, rather than direct ligand clashing, by shifting the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with orthosteric ligand binding. Crystal structures reveal different cobinding mechanisms including alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent inhibitor binding pose. Our findings highlight the significant flexibility of the PPARγ orthosteric pocket, its ability to accommodate multiple ligands, and demonstrate that GW9662 and T0070907 should not be used as chemical tools to inhibit ligand binding to PPARγ.
-
- 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.
