Structure of a AAA+ unfoldase in the process of unfolding substrate
- The Hospital for Sick Children Research Institute, Canada
- University of Toronto, Canada
Figures
Figure 1 with 1 supplement
ΔN-VAT unfolds other copies of ΔN-VAT.
(a) Cartoon showing VAT (purple ring) working in concert with the 20S proteasome (blue and pink rings) to degrade protein substrates (yellow). (b) An SDS-PAGE gel showing that both ΔN-VAT and …
Figure 1—figure supplement 1
Domain arrangement and oligomeric structure of VAT.
Cartoon representation of the VAT primary structure (a) protomer structure (b) and hexamer structure (c). In (c) the stacked ring structure is shown with the AAA+ nucleotide binding domains colored …
Figure 2 with 2 supplements
Structure of six-fold symmetric ΔN-VAT with bound ATPγS.
(a) Density map and atomic model for ATPγS-bound ΔN-VAT at 3.9 Å resolution. A central pore runs through the middle of the complex. Scale bar, 25 Å. (b) Density map and model for a single protomer …
Figure 2—figure supplement 1
Cryo-EM map calculation and validation.
(a) Fourier shell correlation (FSC) curve for the stacked-ring conformation of ΔN-VAT after a gold-standard refinement (resolutions reported at FSC = 0.143). FSC curves were calculated after masking …
Figure 2—figure supplement 2
Pore loops of VAT.
Compared to the stacked-ring conformation (left) and the split-ring conformation determined previously (Huang et al., 2016) (right), the channel through the lumen of the substrate-engaged state of …
Figure 3 with 2 supplements
Substrate engagement by ΔN-VAT.
(a) Cut-away side view of the density map and model for ΔN-VAT with substrate bound. The substrate complex is shown unsharpened in red above the dashed line. Density for the substrate-gripping loop …
Figure 3—figure supplement 1
Ab initio classification to separate the six-fold symmetric and substrate-engaged states of VAT.
(a) 3D maps from the two classes throughout the ab initio classification scheme. At iteration ~500, the low-resolution shape of the substrate is apparent in the substrate-bound class (red arrows) …
Figure 3—figure supplement 2
Symmetry breaking and seam formation in the AAA+ rings of substrate-engaged VAT.
(a) Cartoon showing the helical arrangement of AAA+ subunits bridged by a single subunit at the seam (gold). (b) Views of the different types of protomer-protomer interfaces at the seam (left) and …
Figure 4
Model for substrate translocation by VAT.
(a) The substrate-bound structure of ΔN-VAT suggests that the enzyme is processive, with a single subunit (green outline) that binds substrate at the lowest position (step 1) releasing as it becomes …
Author response image 1
Six-fold symmetric △N-VAT map filtered to 3.9, 4.8, and 7.0 Å resolution:
https://doi.org/10.7554/eLife.25754.016Videos
Video 1
Conformational changes in VAT upon substrate binding.
Interpolation between the stacked-ring and substrate-bound states of VAT, shown as ribbon diagrams (left) and Cα positions for the pore loop residues (right). The complex is viewed ~15° from the …
Video 2
Conformational changes in VAT protomers upon substrate binding.
Interpolation between the stacked-ring and substrate-bound states of VAT, shown as a ribbon diagram for two VAT protomers. Substrate is shown in red. Pore loop residues are indicated in green. Scale …
Video 3
The hand-over-hand model for substrate translocation.
Interpolation between states where each subunit sequentially goes through the different possible positions in the helix. Each promoter’s pore loop binds a section of substrate at the top of the …
Additional files
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Supplementary file 1
Cryo-EM data acquisition, processing, and atomic model statistics.
- https://doi.org/10.7554/eLife.25754.015
