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Structural characterization of full-length NSF and 20S particles

Nature Structural & Molecular Biology volume 19, pages 268–275 (2012)Cite this article

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Abstract

The 20S particle, which is composed of the N-ethylmaleimide–sensitive factor (NSF), soluble NSF attachment proteins (SNAPs) and the SNAP receptor (SNARE) complex, has an essential role in intracellular vesicle fusion events. Using single-particle cryo-EM and negative stain EM, we reconstructed four related three-dimensional structures: Chinese hamster NSF hexamer in the ATPγS, ADP-AlFx and ADP states, and the 20S particle. These structures reveal a parallel arrangement between the D1 and D2 domains of the hexameric NSF and characterize the nucleotide-dependent conformational changes in NSF. The structure of the 20S particle shows that it holds the SNARE complex at two interaction interfaces around the C terminus and N-terminal half of the SNARE complex, respectively. These findings provide insight into the molecular mechanism underlying disassembly of the SNARE complex by NSF.

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Figure 1: Sample preparation and negative stain EM of NSF in different nucleotide states.
Figure 2: Cryo-EM structure of NSF in the ADP-AlFx state.
Figure 3: Cryo-EM structure of NSF in the ADP state.
Figure 4: Comparison of D2 and D1 structures in the ADP-AlFx and ADP states.
Figure 5: Biochemical analyses of N domain movement and substrate binding activity.
Figure 6: The 3D reconstruction of 20S particles.

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Acknowledgements

We are grateful to E.R. Chapman (University of Wisconsin, Madison), J. Rizo (University of Texas Southwestern Medical Center) for providing plasmids. We thank F. Zhang, N. Gao, J.-W. Wu, N. Yan, J. Wang and L. Cheng for helpful discussions. We also thank J. Lei for setting up a semiautomated data collection program and Y. Wu for providing electron microscope for our use at the early stage of this work. This work was supported by the National Basic Research Program of China (2011CB910500/2010CB833706/2010CB912400) and the National Natural Science Foundation of China (30830028).

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Author notes

  1. Jiansen Jiang

    Present address: Present address: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA.,

  2. Lei-Fu Chang, Song Chen and Cui-Cui Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing, China

    Lei-Fu Chang, Song Chen, Cui-Cui Liu, Xijiang Pan, Jiansen Jiang, Xiao-Chen Bai, Xin Xie & Sen-Fang Sui

  2. Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Lei-Fu Chang, Song Chen, Cui-Cui Liu, Xijiang Pan, Jiansen Jiang, Xiao-Chen Bai, Xin Xie, Hong-Wei Wang & Sen-Fang Sui

  3. Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

    Hong-Wei Wang

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Contributions

L.-F.C., S.C. and C.-C.L. conducted the experiments and analyzed the data with the help of X.P., J.J., X.-C.B. and X.X.; L.-F.C. prepared the figures; H.-W.W. commented on the experiments; J.J. and H.-W.W edited the manuscript; L.-F.C., S.C. and S.-F.S. planned the experiments and prepared the manuscript; S.-F.S. coordinated the project.

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Correspondence to Sen-Fang Sui.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Tables 1 and 2, and Supplementary Methods (PDF 1827 kb)

Supplementary Movie 1

This movie shows the cryo-EM map of a NSF protomer in ADP-AlFx state. Crystal structures of D1 and D2 were flexibly fitted as in Figure 2a. (MOV 3514 kb)

Supplementary Movie 2

This movie shows the cryo-EM map of a NSF protomer in ADP state. Crystal structures of D1 and D2 were flexibly fitted as in Figure 3a. (MOV 3198 kb)

Supplementary Movie 3

This movie shows the linear morphing between the D1 hexamer (ribbon diagram of flexibly fitted structures) in the ADP-AlFx state and in the ADP state as shown in Figure 4d, which was generated by “morph conformations” program in Chimera. (MOV 1804 kb)

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Chang, LF., Chen, S., Liu, CC. et al. Structural characterization of full-length NSF and 20S particles. Nat Struct Mol Biol 19, 268–275 (2012). https://doi.org/10.1038/nsmb.2237

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