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

G蛋白偶联受体 低温电子显微 跨膜结构域 计算生物学 生物物理学 跨膜蛋白 生物 化学 受体 生物化学
作者
Jun Xu,Geng Chen,Haoqing Wang,Sheng Cao,Jie Heng,Xavier Deupí,Yang Du,Brian K. Kobilka
出处
期刊:Proceedings of the National Academy of Sciences of the United States of America [National Academy of Sciences]
卷期号:121 (48) 被引量:1
标识
DOI:10.1073/pnas.2414544121
摘要

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.
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