Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

  1. Brandon Wey-Hung Liauw
  2. Arash Foroutan
  3. Michael R Schamber
  4. Weifeng Lu
  5. Hamid Samareh Afsari  Is a corresponding author
  6. Reza Vafabakhsh  Is a corresponding author
  1. Northwestern University, United States
  2. Boehringer Ingelheim Pharmaceuticals, Inc, United States

Abstract

Activation of G protein-coupled receptors (GPCRs) is an allosteric process. It involves conformational coupling between the orthosteric ligand binding site and the G protein binding site. Factors that bind at non-cognate ligand binding sites to alter the allosteric activation process are classified as allosteric modulators and represent a promising class of therapeutics with distinct modes of binding and action. For many receptors, how modulation of signaling is represented at the structural level is unclear. Here, we developed FRET sensors to quantify receptor modulation at each of the three structural domains of metabotropic glutamate receptor 2 (mGluR2). We identified the conformational fingerprint for several allosteric modulators in live cells. This approach enabled us to derive a receptor-centric representation of allosteric modulation and to correlate structural modulation to the standard signaling modulation metrics. Single-molecule FRET analysis revealed that a NAM increases the occupancy of one of the intermediate states while a PAM increases the occupancy of the active state. Moreover, we found that the effect of allosteric modulators on the receptor dynamics is complex and depend on the orthosteric ligand. Collectively, our findings provide a structural mechanism of allosteric modulation in mGluR2 and suggest possible strategies for design of future modulators.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Accompanying source data is provided for figures 1-4 and tables 1-3. The PDB accession codes for human mGluR2 structures used are 7MTS, 7MTR, 7E9G, 7EPE, and 7EPF.

The following previously published data sets were used

Article and author information

Author details

  1. Brandon Wey-Hung Liauw

    Department of Molecular Biosciences, Northwestern University, Evanston, United States
    Competing interests
    No competing interests declared.
  2. Arash Foroutan

    Department of Molecular Biosciences, Northwestern University, Evanston, United States
    Competing interests
    No competing interests declared.
  3. Michael R Schamber

    Department of Molecular Biosciences, Northwestern University, Evanston, United States
    Competing interests
    No competing interests declared.
  4. Weifeng Lu

    Department of Molecular Biosciences, Northwestern University, Evanston, United States
    Competing interests
    No competing interests declared.
  5. Hamid Samareh Afsari

    Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, United States
    For correspondence
    hamid.samareh_afsari@boehringer-ingelheim.com
    Competing interests
    Hamid Samareh Afsari, is affiliated with Boehringer Ingelheim Pharma GmbH & Co. The author has no financial interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5839-4765
  6. Reza Vafabakhsh

    Department of Molecular Biosciences, Northwestern University, Evanston, United States
    For correspondence
    reza.vafabakhsh@northwestern.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8384-3203

Funding

NIGMS (R01GM140272)

  • Reza Vafabakhsh

NIGMS (T32GM-008061)

  • Brandon Wey-Hung Liauw

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

Reviewing Editor

  1. Marcel P Goldschen-Ohm, University of Texas at Austin, United States

Publication history

  1. Received: March 26, 2022
  2. Preprint posted: April 28, 2022 (view preprint)
  3. Accepted: June 30, 2022
  4. Accepted Manuscript published: July 1, 2022 (version 1)
  5. Version of Record published: July 20, 2022 (version 2)

Copyright

© 2022, Liauw 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

  • 1,464
    Page views
  • 379
    Downloads
  • 3
    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. Brandon Wey-Hung Liauw
  2. Arash Foroutan
  3. Michael R Schamber
  4. Weifeng Lu
  5. Hamid Samareh Afsari
  6. Reza Vafabakhsh
(2022)
Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2
eLife 11:e78982.
https://doi.org/10.7554/eLife.78982

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Maura Greiser, Mariusz Karbowski ... Liron Boyman
    Research Article

    Mitochondrial ATP production in cardiac ventricular myocytes must be continually adjusted to rapidly replenish the ATP consumed by the working heart. Two systems are known to be critical in this regulation: mitochondrial matrix Ca2+ ([Ca2+]m) and blood flow that is tuned by local ventricular myocyte metabolic signaling. However, these two regulatory systems do not fully account for the physiological range of ATP consumption observed. We report here on the identity, location, and signaling cascade of a third regulatory system -- CO2/bicarbonate. CO2 is generated in the mitochondrial matrix as a metabolic waste product of the oxidation of nutrients that powers ATP production. It is a lipid soluble gas that rapidly permeates the inner mitochondrial membrane (IMM) and produces bicarbonate (HCO3-) in a reaction accelerated by carbonic anhydrase (CA). The bicarbonate level is tracked physiologically by a bicarbonate-activated adenylyl cyclase, soluble adenylyl cyclase (sAC). Using structural Airyscan super-resolution imaging and functional measurements we find that sAC is primarily inside the mitochondria of ventricular myocytes where it generates cAMP when activated by HCO3-. Our data strongly suggest that ATP production in these mitochondria is regulated by this cAMP signaling cascade operating within the inter-membrane space (IMS) by activating local EPAC1 (Exchange Protein directly Activated by cAMP) which turns on Rap1 (Ras-related protein 1). Thus, mitochondrial ATP production is shown to be increased by bicarbonate-triggered sAC signaling through Rap1. Additional evidence is presented indicating that the cAMP signaling itself does not occur directly in the matrix. We also show that this third signaling process involving bicarbonate and sAC activates the cardiac mitochondrial ATP production machinery by working independently of, yet in conjunction with, [Ca2+]m-dependent ATP production to meet the energy needs of cellular activity in both health and disease. We propose that the bicarbonate and calcium signaling arms function in a resonant or complementary manner to match mitochondrial ATP production to the full range of energy consumption in cardiac ventricular myocytes in health and disease.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Saif Khan, Cornelius Gati
    Insight

    A complex interplay between structure, conformational dynamics and pharmacology defines distant regulation of G protein-coupled receptors.