Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2

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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

(2021) CryoEM Structure of mGlu2 - Gi Complex
PBD, 7MTS.

https://www.rcsb.org/structure/7MTS
(2021) CryoEM Structure of Full-Length mGlu2 Bound to Ago-PAM ADX55164 and Glutamate
PDB, 7MTR.

https://www.rcsb.org/structure/7MTR
1. Lin S
2. Han S
3. Zhao Q
4. Wu B
(2021) Cryo-EM structure of Gi-bound metabotropic glutamate receptor mGlu2
PDB, 7E9G.

https://www.rcsb.org/structure/7E9G
1. Du J
2. Wang D
3. Lin S
4. Han S
5. Wu B
6. Zhao Q
(2021) Crystal structure of mGlu2 bound to NAM563
PDB, 7EPE.

https://www.rcsb.org/structure/7EPE
1. Du J
2. Wang D
3. Lin S
4. Han S
5. Wu B
6. Zhao Q
(2021) Crystal structure of mGlu2 bound to NAM597
PDB, 7EPF.

https://www.rcsb.org/structure/7EPF

Article and author information

Author details

Brandon Wey-Hung Liauw

Department of Molecular Biosciences, Northwestern University, Evanston, United States

Competing interests
No competing interests declared.
Arash Foroutan

Department of Molecular Biosciences, Northwestern University, Evanston, United States

Competing interests
No competing interests declared.
Michael R Schamber

Department of Molecular Biosciences, Northwestern University, Evanston, United States

Competing interests
No competing interests declared.
Weifeng Lu

Department of Molecular Biosciences, Northwestern University, Evanston, United States

Competing interests
No competing interests declared.
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..

"This ORCID iD identifies the author of this article:" 0000-0002-5839-4765
Reza Vafabakhsh

Department of Molecular Biosciences, Northwestern University, Evanston, United States

For correspondence
reza.vafabakhsh@northwestern.edu

Competing interests
No competing interests declared.

"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.

Copyright

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.