Cryo-EM structure of CYP2C9 reveals a dimer-of-trimers assembly

Abstract

Human cytochrome P450 2C9 (CYP2C9) is a hepatic microsomal enzyme involved in the oxidative metabolism of clinically important drugs, but the structural organization of its oligomeric assemblies outside crystallographic packing environments remains poorly understood. Here, we report the cryo-EM structure of human CYP2C9 determined under aqueous, membrane-free conditions at 3.31 Angstrom resolution. The structure reveals a C2-symmetric hexameric assembly organized as a dimer of trimers. Individual protomers retain the conserved P450 fold and heme-binding architecture observed in previously reported crystal structures, indicating that assembly formation does not substantially perturb the catalytic core. The hexamer is stabilized by defined intra-trimer interfaces involving the N-terminal region and residues around Trp212 and Phe482, together with inter-trimer interfaces involving Leu71 and the 220-227 loop. These interfaces are distinct from the crystal packing contacts observed in CYP2C9 crystal structures, demonstrating that the assembly is not a simple recapitulation of crystallographic packing. Notably, the inter-trimer interface is located near the FG-loop-containing surface previously implicated in membrane association. This suggests that the observed hexamer may represent a membrane-free association of two trimers through membrane-related surfaces, whereas the trimeric arrangement itself may be compatible with membrane-associated organization. The structure therefore provides a framework for investigating how trimer formation, membrane interaction and local conformational changes in the FG-loop region may influence CYP2C9 function.

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BibTeX RIS

BibTeX

@misc{tanino2026cryoem,
  title = {{Cryo-EM structure of CYP2C9 reveals a dimer-of-trimers assembly}},
  author = {H. Tanino and H. Tsujino and T. Nakao and C. Oie and F. Makino and T. Miyata and K. Kasai and K. Namba and T. Inoue},
  howpublished = {bioRxiv},
  year = {2026},
  month = aug,
  doi = {10.64898/2026.08.30.747968},
  url = {https://www.biorxiv.org/content/10.64898/2026.08.30.747968v1},
  note = {CC BY-NC-ND},
}

RIS

TY  - UNPB
TI  - Cryo-EM structure of CYP2C9 reveals a dimer-of-trimers assembly
AU  - H. Tanino
AU  - H. Tsujino
AU  - T. Nakao
AU  - C. Oie
AU  - F. Makino
AU  - T. Miyata
AU  - K. Kasai
AU  - K. Namba
AU  - T. Inoue
PY  - 2026
DA  - 2026/08/30
PB  - bioRxiv
DO  - 10.64898/2026.08.30.747968
UR  - https://www.biorxiv.org/content/10.64898/2026.08.30.747968v1
AB  - Human cytochrome P450 2C9 (CYP2C9) is a hepatic microsomal enzyme involved in the oxidative metabolism of clinically important drugs, but the structural organization of its oligomeric assemblies outside crystallographic packing environments remains poorly understood. Here, we report the cryo-EM structure of human CYP2C9 determined under aqueous, membrane-free conditions at 3.31 Angstrom resolution. The structure reveals a C2-symmetric hexameric assembly organized as a dimer of trimers. Individual protomers retain the conserved P450 fold and heme-binding architecture observed in previously reported crystal structures, indicating that assembly formation does not substantially perturb the catalytic core. The hexamer is stabilized by defined intra-trimer interfaces involving the N-terminal region and residues around Trp212 and Phe482, together with inter-trimer interfaces involving Leu71 and the 220-227 loop. These interfaces are distinct from the crystal packing contacts observed in CYP2C9 crystal structures, demonstrating that the assembly is not a simple recapitulation of crystallographic packing. Notably, the inter-trimer interface is located near the FG-loop-containing surface previously implicated in membrane association. This suggests that the observed hexamer may represent a membrane-free association of two trimers through membrane-related surfaces, whereas the trimeric arrangement itself may be compatible with membrane-associated organization. The structure therefore provides a framework for investigating how trimer formation, membrane interaction and local conformational changes in the FG-loop region may influence CYP2C9 function.
ER  -

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