Multiple allosteric sites allow for synergistic enhancement of GPCR signaling
8 affiliations
- 1State Key Laboratory of Drug Research, State Key Laboratory of Chemical Biology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
- 2University of Chinese Academy of Sciences, Beijing 100049, China.
- 3Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
- 4Computational Chemistry and Molecular Biophysics Section, National Institute of Drug Abuse, National Institutes of Health, Baltimore, MD, USA.
- 5Department of Pharmaceutical and Artificial-Intelligence Sciences, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China.
- 6UNC Eshelman School of Pharmacy, Chapel Hill, NC, USA.
- 7Artificial Intelligence Clinical Research Center for drug discovery, Shanghai Key Laboratory of Flexible Medical Robotics, Tongren Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China.
- 8Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China.
Abstract
Allosteric modulation of G protein-coupled receptors (GPCRs) offers promising therapeutic opportunities. Although multiple allosteric binding sites have been identified in GPCRs, whether and how these spatially distinct sites cooperate to regulate receptor activation remain poorly understood. Here, we elucidate the molecular mechanism of cooperative multisite allostery in the dopamine D1 receptor (D1R). We developed two novel D1R positive allosteric modulators (PAMs), UNC9815 and UNC10062, and used cryo-electron microscopy to structurally identify two previously uncharacterized D1R allosteric sites occupied by UNC9815/UNC10062 and another D1R PAM, BMS-A1. Both sites localize to the transmembrane helix (TM) 1 to 7 interface as vertically adjacent allosteric pockets organized in a spatially complementary manner. Our structures demonstrate the simultaneous binding of the chemically distinct PAMs (LY3154207, BMS-A1, and UNC9815/UNC10062) in three nonoverlapping pockets. Concurrent engagement of all three allosteric sites produces pronounced functional cooperativity, increasing dopamine potency by >1000-fold, thereby demonstrating that coordinated multisite modulation can achieve substantial pharmacological amplification beyond the limits imposed by single PAM administration. Molecular dynamics (MD) simulations revealed that multisite PAM engagement restricts intracellular loop 3 dynamics and stabilizes the TM7-NPxxY activation motif, effects not achieved by binding of LY3154207 alone, thereby biasing the receptor toward a more stabilized active conformation, and may also improve agonist potency by stabilizing extracellular loop 2. Together, these findings establish the structural framework for cooperative multisite allostery in a GPCR, revealing how distributed allosteric sites act in concert to amplify receptor activation and providing a conceptual foundation for developing innovative GPCR allosteric therapeutics.
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BibTeX
@article{wang2026multiple,
title = {{Multiple allosteric sites allow for synergistic enhancement of GPCR signaling}},
author = {Kai Wang and Snezana T. Dimova and John N. Hanson and Abhishek Thakur and Youwei Xu and Yini Liu and Amy E. Moritz and Alexander Goldberg and Bing Xie and Jayachandra Rayadurgam and Feijun Wang and Ashley N. Nilson and Kathryn D. Luderman and R. Benjamin Free and Wen Hu and Jian Zhang and H. Eric Xu and Kevin J. Frankowski and Yue Wang and Lei Shi and David R. Sibley and Youwen Zhuang},
journal = {Science Advances},
year = {2026},
month = oct,
doi = {10.1126/sciadv.aeh3401},
url = {https://www.science.org/doi/10.1126/sciadv.aeh3401},
note = {CC BY},
}
RIS
TY - JOUR
TI - Multiple allosteric sites allow for synergistic enhancement of GPCR signaling
AU - Kai Wang
AU - Snezana T. Dimova
AU - John N. Hanson
AU - Abhishek Thakur
AU - Youwei Xu
AU - Yini Liu
AU - Amy E. Moritz
AU - Alexander Goldberg
AU - Bing Xie
AU - Jayachandra Rayadurgam
AU - Feijun Wang
AU - Ashley N. Nilson
AU - Kathryn D. Luderman
AU - R. Benjamin Free
AU - Wen Hu
AU - Jian Zhang
AU - H. Eric Xu
AU - Kevin J. Frankowski
AU - Yue Wang
AU - Lei Shi
AU - David R. Sibley
AU - Youwen Zhuang
PY - 2026
DA - 2026/10/21
JO - Science Advances
DO - 10.1126/sciadv.aeh3401
UR - https://www.science.org/doi/10.1126/sciadv.aeh3401
AB - Allosteric modulation of G protein-coupled receptors (GPCRs) offers promising therapeutic opportunities. Although multiple allosteric binding sites have been identified in GPCRs, whether and how these spatially distinct sites cooperate to regulate receptor activation remain poorly understood. Here, we elucidate the molecular mechanism of cooperative multisite allostery in the dopamine D1 receptor (D1R). We developed two novel D1R positive allosteric modulators (PAMs), UNC9815 and UNC10062, and used cryo-electron microscopy to structurally identify two previously uncharacterized D1R allosteric sites occupied by UNC9815/UNC10062 and another D1R PAM, BMS-A1. Both sites localize to the transmembrane helix (TM) 1 to 7 interface as vertically adjacent allosteric pockets organized in a spatially complementary manner. Our structures demonstrate the simultaneous binding of the chemically distinct PAMs (LY3154207, BMS-A1, and UNC9815/UNC10062) in three nonoverlapping pockets. Concurrent engagement of all three allosteric sites produces pronounced functional cooperativity, increasing dopamine potency by >1000-fold, thereby demonstrating that coordinated multisite modulation can achieve substantial pharmacological amplification beyond the limits imposed by single PAM administration. Molecular dynamics (MD) simulations revealed that multisite PAM engagement restricts intracellular loop 3 dynamics and stabilizes the TM7-NPxxY activation motif, effects not achieved by binding of LY3154207 alone, thereby biasing the receptor toward a more stabilized active conformation, and may also improve agonist potency by stabilizing extracellular loop 2. Together, these findings establish the structural framework for cooperative multisite allostery in a GPCR, revealing how distributed allosteric sites act in concert to amplify receptor activation and providing a conceptual foundation for developing innovative GPCR allosteric therapeutics.
SN - 2375-2548
ER -