Calcineurin-fusion facilitates cryo-EM structure determination of a Family A GPCR.

Autor: Xu J; Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305., Chen G; Kobilka Institute of Innovative Drug Discovery, School of Medicine, Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China., Wang H; Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305., Cao S; Kobilka Institute of Innovative Drug Discovery, School of Medicine, Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China., Heng J; Beijing Advanced Innovation Center for Structural Biology, School of Medicine, Tsinghua University, Beijing 100084, China., Deupi X; Condensed Matter Theory Group, Division of Scientific Computing, Theory, and Data, Paul Scherrer Institute, 5232 Villigen, Switzerland.; Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, 5232 Villigen, Switzerland.; Swiss Institute of Bioinformatics, Lausanne, Switzerland., Du Y; Kobilka Institute of Innovative Drug Discovery, School of Medicine, Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China., Kobilka BK; Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305.
Jazyk: angličtina
Zdroj: Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2024 Nov 26; Vol. 121 (48), pp. e2414544121. Date of Electronic Publication: 2024 Nov 20.
DOI: 10.1073/pnas.2414544121
Abstrakt: Advances in singe-particle cryo-electron microscopy (cryo-EM) have made it possible to solve the structures of numerous Family A and Family B G protein-coupled receptors (GPCRs) in complex with G proteins and arrestins, as well as several Family C GPCRs. Determination of these structures has been facilitated by the presence of large extramembrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during the processing of the cryo-EM data. In contrast, determination of the inactive state structure of Family A GPCRs is more challenging due to the relatively small size of the seven transmembrane domain (7TM) and to the surrounding detergent micelle that, in the absence of other features, make particle alignment impossible. Here, we describe an alternative protein engineering strategy where the heterodimeric protein calcineurin is fused to a GPCR by three points of attachment, the cytoplasmic ends of TM5, TM6, and TM7. This three-point attachment provides a more rigid link with the GPCR transmembrane domain that facilitates particle alignment during data processing, allowing us to determine the structures of the β 2 adrenergic receptor (β 2 AR) in the apo, antagonist-bound, and agonist-bound states. We expect that this fusion strategy may have broad application in cryo-EM structural determination of other Family A GPCRs.
Competing Interests: Competing interests statement:Brian Kobilka is a co-founder of an consultant for ConfometRx. Confometrx has no financial interest in this paper. Brian Kobilka is a co-founder of ConfmetRx.
Databáze: MEDLINE
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