Abstract
The receptor tyrosine kinase ROS1 plays essential roles in cell growth and sperm maturation, yet its activation mechanism has remained poorly understood. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of chicken ROS1 in its ligand-free form, in complex with its ligand NEL, and with the ligand/co-ligand complex NEL/NICOL. Unliganded ROS1 adopts an arc-shaped conformation. The interaction between NEL and ROS1 is mediated by the VWC2 domain of NEL and the β1 domain of ROS1. Binding of NICOL to the coiled-coil domain of NEL stabilizes NEL into a batwing-shaped asymmetric dimer, which can recruit only one ROS1 molecule due to steric hindrance. Structural analyses and biochemical results suggest that the 2:1 NEL/NICOL complexes further oligomerize through LamG–VWC4 domain interactions, facilitating the clustering of multiple ROS1 for its activation. Functional assays confirm that both NICOL and the multimerization of NEL/NICOL complexes are required for robust ROS1 signaling. Our findings establish NICOL as a critical co-ligand for ROS1 and suggest a distinct ligand-driven oligomerization mechanism for ROS1 activation.
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Data availability
All reagents generated in this study are available with a complete Materials Transfer Agreement. All cryo-EM maps and models reported in this work have been deposited into EMDB/PDB database, under the entry ID: ligand-free-ROS1 head region EMDB EMD-71042; PDB: 9OYZ), ligand-free-ROS1 leg region EMDB: EMD-71047; PDB 9OZ1), ligand-free-ROS1 complete model EMDB EMD-71059; PDB: 9OZI), 1:1 cROS1/cNEL complex EMDB EMD-71049; PDB 9OZ6), 2:1 cNEL/hNICOL complex EMDB EMD-71051; PDB 9OZ8), 1:2:1 cROS1/cNEL/hNICOL holo-complex conformation 1 EMDB EMD-71057; PDB 9OZC), and 1:2:1 cROS1/cNEL/hNICOL holo-complex conformation 1 EMDB EMD-71058; PDB 9OZH). The source data for Figs. 2e, 5c, and Supplementary Fig. S7b, S7c, S8, S9a, S9b, S13b, and S13c are provided in a Source Data file. Source data are provided with this paper.
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Acknowledgements
Cryo-EM data were collected at the University of Texas Southwestern Medical Center (UTSW) Cryo-Electron Microscopy Facility, funded in part by the Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Support Award RP170644. We thank Dr. Stoddard for facility access. We thank Dr. Chad A. Brautigam and Dr. Shih-Chia (Scott) Tso for the assistant of SEC-MALS experiments. We thank the Structural Biology Laboratory at UTSW for equipment use. This work is supported in part by grants from the National Institutes of Health (R35GM130289 to X.Z. and R35GM156386 to X.-c.B.), the Welch foundation (I-1702 to X.Z. and I-1944 to X.-c.B.). X.-c.B. and X.Z. are Virginia Murchison Linthicum Scholars in Medical Research at UTSW.
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X.Z. and X.-C.B. designed and supervised research; W.A. designed and performed biochemical and cell biological experiments. W.A., X.Z., and X.-C.B. performed structural biology experiments, X.Z. and X.-C.B. built and refined structural models. All the authors analyzed data; all the authors contributed to the writing, editing and reviewing of the manuscript.
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An, W., Zhang, X. & Bai, Xc. Structural insights into the activation of the chicken ROS1 receptor by the NEL/NICOL ligand complex. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69942-8
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DOI: https://doi.org/10.1038/s41467-026-69942-8
