Abstract
Among the distinct adaptor protein (AP) complexes, AP-4 primarily functions as a non-clathrin-coated vesicle machinery essential for intracellular membrane trafficking. ARF1 is a master regulator of AP-4 membrane recruitment, but the underlying mechanism remains elusive. Here, we present the cryo-EM structures of soluble AP-4 and the AP-4/ARF1 complex. Unexpectedly, AP-4 adopts a dynamic equilibrium between closed and open conformations, caused by loose contacts between its medium subunit and central core. ARF1 binding induces only subtle changes in AP-4, which retains its conformational equilibrium. Mutations at the AP-4/ARF1 interface disrupt complex formation and impair ARF1-dependent membrane recruitment. Efficient membrane recruitment of AP-4 likely requires the s…
Abstract
Among the distinct adaptor protein (AP) complexes, AP-4 primarily functions as a non-clathrin-coated vesicle machinery essential for intracellular membrane trafficking. ARF1 is a master regulator of AP-4 membrane recruitment, but the underlying mechanism remains elusive. Here, we present the cryo-EM structures of soluble AP-4 and the AP-4/ARF1 complex. Unexpectedly, AP-4 adopts a dynamic equilibrium between closed and open conformations, caused by loose contacts between its medium subunit and central core. ARF1 binding induces only subtle changes in AP-4, which retains its conformational equilibrium. Mutations at the AP-4/ARF1 interface disrupt complex formation and impair ARF1-dependent membrane recruitment. Efficient membrane recruitment of AP-4 likely requires the synergistic engagement of ARF1 and cargoes. Disrupting the conformational flexibility of AP-4 interferes with this synergistic effect and compromises AP-4-mediated membrane trafficking. Our findings may redefine AP-4 as a conformationally dynamic complex modulated by cooperative interactions, providing insights into neurodevelopmental disorders associated with AP-4 dysfunction.
Data availability
The atomic coordinates of the AP-4 core complex have been deposited in the protein data bank with the accession codes: 9U9I (μ4-CTD-undocked) and 9U9J (μ4-CTD-docked). The atomic coordinates of the AP-4/ARF1 complex have been deposited in the protein data bank with the accession codes: 9U9R (μ4-CTD-undocked) and 9U9S (μ4-CTD-docked). The three-dimensional cryo-EM density maps of the AP-4 core complex have been deposited in the Electron Microscopy Data Bank with the accession codes: EMD-63965 (μ4-CTD-undocked) and EMD-63966 (μ4-CTD-docked. The three-dimensional cryo-EM density maps of the AP-4/ARF1 complex have been deposited in the Electron Microscopy Data Bank with the accession codes: EMD-63968 (μ4-CTD-undocked) and EMD-63969 (μ4-CTD-docked). The atomic coordinates used for the model building and docking of the AP-4 core and AP-4/ARF1 complexes were downloaded from the Protein Data Bank with the accession codes: 1J2J (ARF1), 1W63 (AP-1 core). The atomic coordinates used for structural comparisons and SWISS-MODEL building were downloaded from the Protein Data Bank with the accession codes: 4HMY, 6DFF, 2VGL, 7R4H, 7RW9, 2JKR, 2XA7, 6QH6, 7P3X, 7P3Y, 7P3Z, 9C5C, 9C5B, 9C58, 6QH5, 3TJZ, 5A1U. The mass spectrometry proteomics data of the AP-4/ARF1 complex have been deposited in the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD072343. The source data underlying Figs. 1b, 3b, c, 4e, g–k, 5c–e and 6g and Supplementary Figs. 8a, b, 10a, b, 11f and 13b are provided as a Source Data file. Source data are provided in this paper.
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Acknowledgements
We thank Xilin Miao, Dr. Jie Ren, Dr. Huaqing Cai and Dr. Junjie Hu for the help on the liposome flotation assay, Dr. Sarah Perrett for the help on the smFRET assay, Kewei Gu for the help on the cellular studies, and Jifeng Wang for the help on mass spectrometry. This work was supported by grants from the Brain Science and Brain-like Intelligence Technology – National Science and Technology Major Project (2022ZD0205800 to Y.Z.), the CAS Project for Young Scientists in Basic Research (YSBR-104 to W.F. and Y.Z.), and the National Natural Science Foundation of China (32571398 to W.F.). We acknowledge the support from the State Key Laboratory of Biomacromolecules and State Key Laboratory of Epigenetic Regulation and Intervention (to S.W.).
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Author notes
These authors contributed equally: Yanghui Wang, Wei Li, Yunlong Qiu, Si Wu.
Authors and Affiliations
State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing, China
Yanghui Wang, Wei Li, Yunlong Qiu, Si Wu, Yan Zhao & Wei Feng 1.
College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
Yanghui Wang, Yunlong Qiu, Yan Zhao & Wei Feng 1.
School of Mathematics, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou, China
Liu Hong
Authors
- Yanghui Wang
- Wei Li
- Yunlong Qiu
- Si Wu
- Liu Hong
- Yan Zhao
- Wei Feng
Contributions
Y.W., W.L., Y.Q., and S.W. conducted experiments and participated in the design and interpretation of the experiments. Specifically, Y.W. and W.L. conducted all biochemical and cellular experiments, Y.W. and Y.Q. performed structural studies, and Y.W. and S.W. performed the smFRET assay. L.H. provided the help on the smFRET data analysis. Y.Z. provided the guidance for the cryo-EM data collection and analysis. W.F. designed all the experiments, interpreted the data and wrote the manuscript.
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Correspondence to Yan Zhao or Wei Feng.
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Wang, Y., Li, W., Qiu, Y. et al. Structural basis for the dynamic conformations of AP-4 and its association with ARF1. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68679-8
Received: 17 April 2025
Accepted: 13 January 2026
Published: 21 January 2026
DOI: https://doi.org/10.1038/s41467-026-68679-8