Research ArticleBIOCHEMISTRY

Mechanism of Vps4 hexamer function revealed by cryo-EM

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Science Advances  14 Apr 2017:
Vol. 3, no. 4, e1700325
DOI: 10.1126/sciadv.1700325
  • Fig. 1 Vps4 hexamers in open and closed conformations.

    (A and B) Vps4 hexamer in the open (A) and closed (B) conformations. Top and middle: Side and top views, respectively, of cryo-EM density maps. Bottom: Top view of structural models. Difference densities (red) corresponding to ATP are shown at the same threshold cutoff. (C) Flexibly fit crystal structure of Vps4 subunit into the corresponding EM density region (subunit D, closed conformation). (D) Structural transitions between the open and closed Vps4 conformers. The conformers are aligned on the basis of subunits B to F (gray) and shown from the side of subunit A in the open (orange) and closed (yellow) conformations.

  • Fig. 2 Subunit interfaces within the Vps4 hexamer.

    (A) Ribbon representation of two neighboring Vps4 subunits with inserts showing details of the three interfaces (I to III). (B) Schematic diagram showing the presence of the three interfaces between different subunit pairs in the two Vps4 hexamer conformers. (C) Only partial ATP-binding sites are formed at the A/B subunit interface of the closed conformer.

  • Fig. 3 Number of subunits required for ATP hydrolysis and ESCRT-III disassembly.

    (A) Titration of Vps4E233Q into one of the Vps4 variants (Vps4WT, Vps4R288A/R289A, Vps4K179A, or Vps4E233Q, all fixed at 0.5 μM). ATPase activity was measured as micromolar inorganic phosphate released per micromolar Vps4 variant per minute. (B) Cartoon illustrating how mixing of Vps4E233Q and Vps4R288A/R289A can produce an active heterohexamer. (C) Electron micrograph of negative-stained filaments of Vps24-Vps2 chimera, before (left) and after incubation with wild-type Vps4 (middle) or Vps4E233Q (right) in the presence of ATP. (D) Disassembly of Vps24-Vps2 filaments by 2.5 μM Vps4WT as Vps4E233Q is titrated into the reaction mixture. Error bars are SD of results from three independent repeats.

  • Fig. 4 Model of Vps4-mediated ESCRT-III disassembly.

    (A) Cryo-EM 3D map and fitted structure model of Vps4 hexamer in the closed conformation, with density corresponding to the stacked pore loop 1 (cyan). The zoom-in view (right) shows the map and model for pore loop 1 densities from the six Vps4 subunits. (B) Comparison of pore loop 1 density segmentation between the open and closed conformations. (C) Model of Vps4-mediated ESCRT-III disassembly. Vps4 forms an open hexamer in the presence of ATP. Binding of one ESCRT-III subunit to the pore loops of subunit A stimulates its ATPase activity. Upon hydrolysis, subunit A dissociates from subunit B and establishes a new contact with subunit F through a closed ring hexamer intermediate. Eventually, a new open Vps4 hexamer is formed, where A repositions by ~33 Å and becomes the new F whereas B becomes the new A. The ESCRT-III subunit bound to A will experience the mechanical force accompanying the conformational change of the subunit in the hexamer.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/4/e1700325/DC1

    fig. S1. Vps4 oligomerizes into a hexamer in the presence of ATP.

    fig. S2. Cryo-EM images of Vps4 oligomer.

    fig. S3. Flow chart of particle classification and 3D map reconstruction.

    fig. S4. Map resolution estimation and projection angle distribution.

    fig. S5. Fitting of the Vps4 hexamer structure into the cryo-EM map.

    fig. S6. Molecular dynamics flexible fitting.

    fig. S7. Comparison of the crystal structure and Vps4 hexamer subunit structures.

    fig. S8. Sequence alignments of Vps4 proteins from S. cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens.

    fig. S9. Residues at subunit interface III are important for Vps4 oligomerization and ATPase activity.

    fig. S10. Structural comparison of Vps4 subunits in the open and closed conformations.

    fig. S11. One wild-type subunit per hexamer is sufficient to maintain full Vps4 hexamer ATPase activity.

    fig. S12. Filament disassembly activity of Vps4.

    movie S1. Molecular dynamic flexible fitting into Vps4 open and closed cryo-EM maps.

    movie S2. Morphing motion between Vps4 open and closed models.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Vps4 oligomerizes into a hexamer in the presence of ATP.
    • fig. S2. Cryo-EM images of Vps4 oligomer.
    • fig. S3. Flow chart of particle classification and 3D map reconstruction.
    • fig. S4. Map resolution estimation and projection angle distribution.
    • fig. S5. Fitting of the Vps4 hexamer structure into the cryo-EM map.
    • fig. S6. Molecular dynamics flexible fitting.
    • fig. S7. Comparison of the crystal structure and Vps4 hexamer subunit structures.
    • fig. S8. Sequence alignments of Vps4 proteins from S. cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens.
    • fig. S9. Residues at subunit interface III are important for Vps4 oligomerization and ATPase activity.
    • fig. S10. Structural comparison of Vps4 subunits in the open and closed conformations.
    • fig. S11. One wild-type subunit per hexamer is sufficient to maintain full Vps4 hexamer ATPase activity.
    • fig. S12. Filament disassembly activity of Vps4.

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1(.mov format). Molecular dynamic flexible fitting into Vps4 open and closed cryo-EM maps.
    • movie S2 (.mov format). Morphing motion between Vps4 open and closed models.

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