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<p>Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαβγ structures, these snapshots primarily capture the fully activated complex. Consequently, the functions of intermediate GPCR-G protein complexes remain elusive. Guided by a conformational landscape visualized via <sup>19</sup>F quantitative NMR and molecular dynamics (MD) simulations, we determined the structure of an intermediate GPCR-mini-Gα<sub>s</sub>βγ complex at 2.6 Å using cryo-EM, by blocking its transition to the fully activated complex. Furthermore, we present direct evidence that the complex at this intermediate state initiates a rate-limited nucleotide exchange before transitioning to the fully activated complex. In this state, BODIPY-GDP/GTP based nucleotide exchange assays further indicated the α-helical domain of the Gα is partially open, allowing it to grasp a nucleotide at a non-canonical binding site, distinct from the canonical nucleotide-binding site. These advances bridge a significant gap in our understanding of the complexity of GPCR signaling.</p>

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Structure and function of a near fully-activated intermediate GPCR-Gαβγ complex

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年1月28日

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  • Maxine Bi,
  • Xudong Wang,
  • Jinan Wang,
  • Jun Xu,
  • Wenkai Sun,
  • Victor Ayo Adediwura,
  • Yinglong Miao,
  • Yifan Cheng,
  • Libin Ye

摘要

Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαβγ structures, these snapshots primarily capture the fully activated complex. Consequently, the functions of intermediate GPCR-G protein complexes remain elusive. Guided by a conformational landscape visualized via 19F quantitative NMR and molecular dynamics (MD) simulations, we determined the structure of an intermediate GPCR-mini-Gαsβγ complex at 2.6 Å using cryo-EM, by blocking its transition to the fully activated complex. Furthermore, we present direct evidence that the complex at this intermediate state initiates a rate-limited nucleotide exchange before transitioning to the fully activated complex. In this state, BODIPY-GDP/GTP based nucleotide exchange assays further indicated the α-helical domain of the Gα is partially open, allowing it to grasp a nucleotide at a non-canonical binding site, distinct from the canonical nucleotide-binding site. These advances bridge a significant gap in our understanding of the complexity of GPCR signaling.