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A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission

Nature Structural & Molecular Biology volume 27, pages 570–580 (2020)Cite this article

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Abstract

The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, multivesicular body biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101–VPS28–VPS37B–MVB12A was determined, revealing an ESCRT-I helical assembly with a 12-molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse-grained (CG) simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor; it has an essential scaffolding and mechanical role in its own right.

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Fig. 1: Structure of the heterotetrameric human ESCRT-I head.
Fig. 2: MVB12A VPF motif is required for interaction with the ESCRT-I head and occludes the TSG101 PTAP motif.
Fig. 3: Helical ESCRT-I head assemblies.
Fig. 4: Helical contacts are required for phagophore closure in autophagy.
Fig. 5: Helical contacts are required for efficient HIV-1 release.
Fig. 6: ESCRT-I assembly from CGMD simulations.

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Data availability

Coordinates and structure factors have been deposited in the Protein Data Bank under accession code PDB 6VME. Uncropped images for Figs. 1c, 2d,f, 4a,e, and 5 and Extended Data Fig. 1 are available in Supplementary Fig. 1; source data for graphs in Fig. 4 are available online.

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Acknowledgements

We thank B. Yang for contributing to early stages of this project, S. Fromm, C. Buffalo, and P. Grob for electron microscopy advice and support, K. Larsen for comments on the manuscript, and J. Briggs for providing immature Gag lattice maps. This work was supported by NIH grants R37 AI112442 (J.H.H.), R01 GM127954 (H.G.W), R01 GM128507 (A.H. and G.A.V.) and F32 AI150477 (A.J.P.). Beamline 8.3.1 at the Advanced Light Source is supported by the National Institutes of Health (R01 GM124149 and P30 GM124169), Plexxikon Inc., and the Integrated Diffraction Analysis Technologies program of the US Department of Energy Office of Biological and Environmental Research. The Advanced Light Source (Berkeley, CA) is a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the US Department of Energy under contract number DE-AC02-05CH11231, Office of Basic Energy Sciences.

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Authors and Affiliations

  1. Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, USA

    Thomas G. Flower, Nicholas Tjahjono, Adam L. Yokom & James H. Hurley

  2. Department of Pediatrics, Pennsylvania State College of Medicine, Hershey, PA, USA

    Yoshinori Takahashi, Xinwen Liang & Hong-Gang Wang

  3. Department of Chemistry, James Franck Institute, and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Arpa Hudait, Alexander J. Pak & Gregory A. Voth

  4. Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Kevin Rose & Fadila Bouamr

  5. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    James H. Hurley

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Contributions

Conceptualization, T.G.F., Y.T., G.A.V., J.H.H.; investigation, T.G.F., Y.T., A.H., K.R., N.T., A.L.Y., X.L.; resources, A.J.P.; supervision, H.-G.W., F.B., G.A.V., J.H.H.; writing − original draft, T.G.F., Y.T., A.H., G.A.V., J.H.H.; writing − review and editing, all authors.

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Correspondence to James H. Hurley.

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J.H.H. is a cofounder of Casma Therapeutics.

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Peer review information Peer reviewer reports are available. Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Effect of specific mutations on ESCRT-I head complex integrity.

Mutant versions of the ESCRT-I head complex were expressed in E. coli. VPS28 and MVB12A subunits were expressed as C-terminal hexahistidine and N-terminal GST fusions respectively. Lysate was incubated with Ni-NTA agarose beads and complex integrity analyzed by SDS-PAGE following multiple washes. The SDS-PAGE image shown is representative of two independent biological repeats. Uncropped image is in Supplementary Fig. 1.

Extended Data Fig. 2 Sequence alignment of VPS28 orthologs.

Secondary structure displayed above the alignment is derived from the human ESCRT-I head structure. Alignment was generated using ClustalW and ESPript.

Extended Data Fig. 3 Helical yeast ESCRT-I head tubes.

The trimeric yeast ESCRT-I head forms helical tubes within a crystal (PDB 2CAZ). The crystal is constructed from a series of laterally stacked tubes where each tube is composed of a single, continuous helix of yeast ESCRT-I head protomers. Vps23, Vps28, Vps37 are colored green, purple and magenta respectively. Tube dimensions are labeled.

Supplementary information

Supplementary Information

Supplementary Fig. 1: uncropped gels from Figs. 1, 2, 4 and 5 and Extended Data Fig. 1.

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Supplementary Video 1

Assembly of ESCRT-I templated by a Gag shell with a 54 nm opening. The color representation is same as described in Fig. 6. For each frame, only the largest ESCRT-I oligomer is shown. We note that for some frames there are two largest oligomers of the same size in which both the oligomers are shown. The membrane is not rendered for visual clarity.

Supplementary Note 1

Details of computational methods

Source data

Source Data Fig. 4

Data for scatter plots

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Flower, T.G., Takahashi, Y., Hudait, A. et al. A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission. Nat Struct Mol Biol 27, 570–580 (2020). https://doi.org/10.1038/s41594-020-0426-4

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