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<p>OsYchF1 belongs to the YchF subfamily in the Obg family of G proteins, and comprises a core G domain, an α-helical domain, and a TGS domain from the N-terminus to the C-terminus. The C2-domain <i>Oryza sativa</i> GTPase-activating protein (OsGAP1) binds to and accelerates the enzyme activity of OsYchF1. The OsYchF1–OsGAP1 module plays a pivotal role in the response of rice to environmental stress. Nonetheless, how OsYchF1 anchors OsGAP1 has remained elusive. Our results demonstrate that OsYchF1 interacts with OsGAP1 through its N-terminal G domain and C-terminal TGS domain, while the inserted helical domain imposes a steric hindrance, as shown by yeast two-hybrid and isothermal titration calorimetry assays. Also, we implemented the AlphaFold2 artificial intelligence system to predict the structure of the OsYchF1–OsGAP1 complex. However, AlphaFold2 could not capture these dynamic conformational changes, likely due to the weak (<i>K</i><sub>D</sub>=102.6 ± 11.7 µM) and transient nature of the OsYchF1-OsGAP1 interaction, which is primarily driven by hydrophobic interactions. Unexpectedly, the protein-protein docking by a fully automated algorithm ClusPro accurately recapitulated both the experimental evidence for G/TGS domain-mediated binding and the known cluster regions on OsGAP1, whereas AlphaFold2 failed to align with these findings.</p>

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From biophysical interaction to structural modeling: bi-terminal G and TGS domains drive rice OsYchF1-OsGAP1 complex formation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年9月29日

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  • Suchang Huang,
  • Ziting Wu,
  • Kuang Wang,
  • Yunchuan Liao,
  • Jiaqiang Zeng,
  • Rui Miao

摘要

OsYchF1 belongs to the YchF subfamily in the Obg family of G proteins, and comprises a core G domain, an α-helical domain, and a TGS domain from the N-terminus to the C-terminus. The C2-domain Oryza sativa GTPase-activating protein (OsGAP1) binds to and accelerates the enzyme activity of OsYchF1. The OsYchF1–OsGAP1 module plays a pivotal role in the response of rice to environmental stress. Nonetheless, how OsYchF1 anchors OsGAP1 has remained elusive. Our results demonstrate that OsYchF1 interacts with OsGAP1 through its N-terminal G domain and C-terminal TGS domain, while the inserted helical domain imposes a steric hindrance, as shown by yeast two-hybrid and isothermal titration calorimetry assays. Also, we implemented the AlphaFold2 artificial intelligence system to predict the structure of the OsYchF1–OsGAP1 complex. However, AlphaFold2 could not capture these dynamic conformational changes, likely due to the weak (KD=102.6 ± 11.7 µM) and transient nature of the OsYchF1-OsGAP1 interaction, which is primarily driven by hydrophobic interactions. Unexpectedly, the protein-protein docking by a fully automated algorithm ClusPro accurately recapitulated both the experimental evidence for G/TGS domain-mediated binding and the known cluster regions on OsGAP1, whereas AlphaFold2 failed to align with these findings.