Intermediate-state-trapped mutants pinpoint G protein-coupled receptor conformational allostery
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1.
California Institute of Technology
Abstract
Understanding the roles of intermediate states in signaling is pivotal to unraveling the activation processes of G protein-coupled receptors (GPCRs). However, the field is still struggling to define these conformational states with sufficient resolution to study their individual functions. Here, we demonstrate the feasibility of enriching the populations of discrete states via conformation-biased mutants. These mutants adopt distinct distributions among five states that lie along the activation pathway of adenosine A2A receptor (A2AR), a class A GPCR. Our study reveals a structurally conserved cation-π lock between transmembrane helix VI (TM6) and Helix8 that regulates cytoplasmic cavity opening as a "gatekeeper" for G protein penetration. A GPCR activation process based on the well-discerned conformational states is thus proposed, allosterically micro-modulated by the cation-π lock and a previously well-defined ionic interaction between TM3 and TM6. Intermediate-state-trapped mutants will also provide useful information in relation to receptor-G protein signal transduction.
Additional Information
This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. This work was supported by the USF startup funds and Internal USF grants (L.Y.), NIH R01HL155532 (W.A.G.), and U.S. Department of Energy (DOE) Laboratory Directed Research and Development funds (C.N.). We thank Amirhossein Mafi for kindly providing MD trajectories of LUF5833-A_(2A)-R-Gαₛβγ for partial agonist LUF5834 docking. We thank Dr. Brian Kobilka and Dr. Jun Xu from Stanford University for providing valuable comments on the manuscript and sending us heterotrimeric Gαₛβγ. We also thank Hiran Malinda Lamabadu Warnakulasurya in assistance of receptor cell culture. Our computations used resources provided by the LANL Institutional Computing Program, which is supported by the U.S. DOE National Nuclear Security Administration under contract DE-AC52-06NA25396, and the Materials and Process Simulation Center (MSC), California Institute of Technology. These authors contributed equally: Xudong Wang, Chris Neale. Contributions. L.Y. conceived and designed the research. X.W. performed the molecular biology work, generated high yield transformants, optimized receptor expression and purification, performed informatic analyses on amino acid sequence conservation and structural similarity, conducted NMR experiments and processed part of NMR data, and conducted radioligand binding assays and SDS-PAGE. C.N. performed MD simulations and bioinformatic analyses. S.K.K. performed docking experiments of LUF5834-A2AR-Gαsβγ. W.A.G. supervised simulation and docking experiments. L.Y. performed NMR and analyzed spectroscopy data. S.K.K., C.N., X.W., and L.Y. prepared the manuscript. All authors read and revised the manuscript. L.Y. supervised the whole project. Data availability. The data that support this study are available from the corresponding authors upon reasonable request. The publicly available datasets with PDB accession codes 2LNL, 2R4S, 2RH1, 2YDO, 3EML, 3PWH, 4AMJ, 4EIY, 4GPO, 4L6R, 6D26, 6GDG, 6W2Y, 7ARO were used in this study for figure preparation and data analyses. Source data underlying figures are provided as a Source Data file. Source data are provided with this paper. The authors declare no competing interests.Attached Files
Published - 41467_2023_Article_36971.pdf
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Supplemental Material - 41467_2023_36971_MOESM4_ESM.xlsx
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Additional details
- PMCID
- PMC10006191
- Eprint ID
- 121748
- Resolver ID
- CaltechAUTHORS:20230607-171926973
- University of South Florida
- R01HL155532
- NIH
- DE-AC52-06NA25396
- Department of Energy (DOE)
- Created
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2023-06-07Created from EPrint's datestamp field
- Updated
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2023-06-08Created from EPrint's last_modified field
- Other Numbering System Name
- WAG
- Other Numbering System Identifier
- 1558