Hsp104, a conserved AAA+ protein disaggregase, promotes survival during cellular stress. Hsp104 remodels amyloids, thereby supporting prion propagation, and disassembles toxic oligomers associated with neurodegenerative diseases. However, a definitive structural mechanism for its disaggregase activity has remained elusive. We determined the cryo-EM structure of wild-type Saccharomyces cerevisiae Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the 12 AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, thus explaining roles in ATP hydrolysis and protein disaggregation. Remarkably, substrate-binding pore loops line the channel in a spiral arrangement optimized for substrate transfer across the AAA+ domains, thereby establishing a continuous path for polypeptide translocation.
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The authors thank J. Smith, Z. March, M. DeSantis, E. Sweeny and J. Lin for reading and discussing the manuscript; S. King for help with figures; and A. Tariq for technical assistance with Hsp104 purification. This work was supported by National Institutes of Health (NIH) grant R01GM099836 (to J.S.). A.L.Y. is supported by an American Heart Association Predoctoral fellowship; M.E.J. is supported by a Target ALS Springboard Fellowship. K.L.M. is supported by an NSF Graduate Research Fellowship (DGE-1321851). J.S. is supported by a Muscular Dystrophy Association Research Award (MDA277268), the Life Extension Foundation, the Packard Center for ALS Research at Johns Hopkins University, and Target ALS. D.R.S. is supported by NIH grants R01GM109896, R01GM077430 and R01GM110001A.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Purified Hsp104 hexamer is active for ATPase function, measured as the release of inorganic phosphate over 5 minutes. Value represents mean ± SEM (n = 3). (b) Purified Hsp104 is active for luciferase disaggregation. Recovered luciferase luminescence determined following incubations with Hsp104 plus (checkered bars) or minus (clear bars) equimolar Hsc70 and Hdj2. Values represent means ± SEM (n = 3). (c) Representative cryo-EM micrograph of Hsp104 hexamers. Scale bar equals 200 Å (d) 2D reference free class averages of Hsp104. The top 50 populated views are shown. (e) Gold standard FSC curve for the un-masked and masked reconstructions of the final model estimated from the FSC=0.143 criterion to be 6.54 Å and 5.64 Å, respectively. (f) 3D plot of the angular distribution for the particles that contributed to the final model. Top and side view orientations are shown with the corresponding 2D projection average. (g) Comparison of reference free class averages and 2D projections of the final 3D reconstruction. Scale bar equals 50 Å. (h) Local resolution of the 3D reconstruction determined by ResMap1 and shown from 5 Å (blue) to 7 Å (red).1 Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat Methods 11, 63–65, doi:10.1038/nmeth.2727 (2014).
(a) ClpB structure labeled for reference, as previously reported1 (PDB: 1QVR), with helices indicated for the NTD (A), NBD1 large (B) and small (C) subdomains, MD (L) and NBD2 large (D) and small (E) subdomains. (b) Hsp104 molecular models for each protomer in the asymmetric hexamer with the segmented density shown at 5σ. Below are enlarged views of the nucleotide pockets with difference maps, generated by subtracting a model with no nucleotide from the cryo-EM map, shown as mesh to visualize density for bound nucleotide. AMPPNP is shown as sticks where density is observed in the pocket for the difference maps.1 Lee, S. et al. The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state. Cell 115, 229–240 (2003).
(a) Overlay of the backbone trace for the protomer NBDs following alignment to the P1 NBD1 large subdomain. RMSD values compared to P1 are shown and identify increasing differences from P2 to P6. (b) Enlarged view of circled region of (a) showing helices E2 and E3 for the 6 aligned protomers, highlighting conformational changes for P1 to P6. (c) Overlay of P1 and P6 protomers that interact at the hexamer interface, showing conformational changes including rotation of the NBD1 and NBD2 small subdomains.
Views showing model with proposed salt bridge interactions between MD-L1 and adjacent NBD1 that bridge the large and small AAA+ subdomains across nucleotide-binding pocket.
Supplementary Figure 5 Additional NBD1 pore loops line the Hsp104 channel and potentially function as substrate-binding sites.
(a) Views of the channel interface with density corresponding to putative NBD1 substrate binding surfaces shown in yellow that include flexible pore loop residues 288-298. Adjacent Tyr pore loop regions are shown as in Figure 5. Hsp104 sequence is shown with conserved charged residues indicated (asterisk).
Supplementary Figures 1–5 (PDF 1010 kb)
Cryo-EM reconstruction of the Hsp104 hexamer colored by domain, as in Figure 1 (MOV 43968 kb)
Cryo-EM map showing a single segmented protomer, P4, and the corresponding molecular model in a ribbons diagram with bound nucleotides as stick models (MOV 30167 kb)
View of the Hsp104 hexamer from inside the channel. Density corresponding to the substrate binding pore loops, colored by protomer as in Figure 2 and 5, is shown arranged in a two turn spiral (MOV 186300 kb)
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Yokom, A., Gates, S., Jackrel, M. et al. Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation. Nat Struct Mol Biol 23, 830–837 (2016). https://doi.org/10.1038/nsmb.3277
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