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

• 成果类型: Article
• 署名作者: Xu, Jun; Chen, Geng; Wang, Haoqing; Cao, Sheng; Heng, Jie; Deupi, Xavier; Du, Yang; Kobilka, Brian K.
• 署名单位: Stanford University; The Chinese University of Hong Kong, Shenzhen; Tsinghua University; Swiss Federal Institutes of Technology Domain; Paul Scherrer Institute; Swiss Federal Institutes of Technology Domain; Paul Scherrer Institute; Swiss Institute of Bioinformatics
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-12527
• DOI: 10.1073/pnas.2414544121
• 发表日期: 2024-11-19
• 关键词: g-protein; crystal-structure; receptor; insights; beta(2)ar

摘要

Advances in singe- particle cryo- electron microscopy (cryo-EM) have made it possible to solve the structures of numerous FamilyA 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 linkwith the GPCR transmembrane domain that facilitates particle alignment during data processing, allowing us to determine the structures of the beta 2 adrenergic receptor (beta 2AR) 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.