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. 2019 Jan 8;116(2):534-539.
doi: 10.1073/pnas.1817752116. Epub 2018 Dec 17.

Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle

Parijat Majumder  1 , Till Rudack  2 , Florian Beck  1 , Radostin Danev  1 , Günter Pfeifer  1 , István Nagy  1 , Wolfgang Baumeister  3
Affiliations

Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle

Parijat Majumder et al. Proc Natl Acad Sci U S A. .

Abstract

Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases.

Keywords: ATPase cycle; PAN; archaea; cryo-EM; proteasome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Architecture of the PAN-proteasome. (A) Cryo-EM reconstruction of double-capped PAN-proteasomes, with applied C2 symmetry. (B) Cutaway view of PSC particle reconstruction. The density maps in both A and B are filtered according to local resolution, with the CP shown in gray and PAN shown in sand. (C) Simplified illustration of a PAN-proteasome top view. The sevenfold symmetric CP is shown in gray, while the different domains of homohexameric PAN are shown in shades of sand. Mismatch of symmetry between PAN and CP leads to an off-axis location of PAN on top of the CP and the axial pores of PAN and CP are clearly misaligned. The domain architecture of PAN is depicted below.
Fig. 2.
Fig. 2.
The PAN-proteasome exists in five rotated spiral-staircase conformations. (A) In the row above, density maps of AAAob from the five conformational states are displayed. For a fixed position of the PAN offset on the CP, the states differ in conformation of their AAA ring, and inclination of the N domain (OB ring and coiled coils). PAN is colored according to protomer, and in each state, the protomer occupying the highest position is indicated by asterisks. AAAob densities are placed in the context of the respective CP (in gray) to emphasize the fixed position of the offset. In the row below, slices through the indicated plane position are displayed. The existence of a split site is clearly visible in the slices and is further highlighted by white arrowheads. (B) Models of different states of the PAN-proteasome are superimposed on the respective PSC densities. In the models, PAN is colored according to protomer, and the CP is in gray. (C) Plot of pore loop height and nucleotide pocket depth for each conformational state (SI Appendix, SI Materials and Methods). Height of the tip of pore helices (α5 helix from each PAN protomer) above the plane of the CP is plotted for every PAN protomer, and is colored coherently. The size of a nucleotide pocket is plotted as an overlay. In all of the states, there is a distinct open pocket (colored white) between the highest and lowest pore helices, while the other pockets (closed) are of similar size, and colored gray.
Fig. 3.
Fig. 3.
Structural basis for intersubunit communication in PAN. (A) A 4.85-Å cryo-EM map of the PAN AAA ring, colored according to constituent protomers. The closed pockets are indicated by asterisks. (Left) Top view of the AAA ring. (Right) The 50°-rotated view shows the spiral-staircase arrangement, highlighting the open and intermediate pockets. (B) Detailed view of the PAN axial channel, showing the arrangement of pore-1 loop tyrosines (Y215). Pore-loop contacts between the I216 backbone O and K214 backbone N are indicated by dashed lines. (C) Detailed view of a closed nucleotide pocket, where the ISS bridge (green) of the left subunit (coral) extends toward the right subunit (violet), establishing contact between F275 and R205. Interaction between the arginine finger (R302) and L269 in the pore-2 helix (α6) brings the α6 helix close to the bound nucleotide. (D) Detailed view of the intermediate pocket, where the ISS bridge (green) of the left subunit (light green) is partially withdrawn, disrupting the interaction between F275 (of the light-green subunit) and R205 (of the pale-yellow subunit). (E) Detailed view of the open pocket, where the ISS bridge of the left subunit (blue) is completely withdrawn, and forms a part of the α6 helix. There is no interaction between F275 and R205 of the adjacent (light-green subunit). In all cases, the arginine fingers (R299 and 302) are consistently colored magenta, and other crucial residues in the nucleotide pockets are colored cyan. The regions depicted in detail in C–E are indicated by boxes on the AAA ring in A, the color of the box matching the outline of the detailed view.
Fig. 4.
Fig. 4.
PAN binding leads to stabilization of the CP gate. Slice (Left) and isosurface (Right) of (A) PAN-bound CP and (B) control CP are shown from the top. In the PAN-bound CP, the axial channel is clear, with finger-like projections surrounding the axial pore. In the control CP, the axial channel appears clear in the isosurface, but is occupied by a smeared density in the slice. (C) Close-up view of the open gate shows gate loops formed by αN-terminal tails. These tails are flexible, and hence missing in the control CP (D). Model showing segments of two adjacent subunits of (E) PAN-bound CP and (F) control CP. The pore of the CP axial channel is lined by P17 reverse turns. In the PAN-bound CP, the residues Y8 and P17 from subunit 2 (pink) interact with D9 and Y26 from subunit 1 (blue), thereby forming an intersubunit quartet. In the control CP, intersubunit contacts are observed between Y26 and P17, although gate loops are missing. (G) In the row above, cutaway views of the PAN-CP interface (filtered to 9 Å) are displayed for the states 1 through 5. The interface densities are placed in the context of the respective PSCs, and colored as in Fig. 2. Asterisks indicate the lowest PAN protomer. The αN-terminal tail that forms a gate contact with PAN is colored according to the PAN protomer it contacts. The boxed regions are magnified in the row below, whereby the gate contacts are indicated by arrowheads of corresponding color.
Fig. 5.
Fig. 5.
Structure-based model for the ATPase cycle in PAN-proteasomes. (A) Cartoon representations of the five ATPase states identified, along with a putative state hypothesized (hypothetical state). In each state, the subunit in the highest and lowest positions is indicated with colored H and L, respectively, and the active (ADP) pocket is indicated by asterisks. (B) Putative scheme of the conformational changes that drive the ATPase cycle around the AAA ring. ATP hydrolysis in the active pocket causes withdrawal of the ISS loop, and upward movement of pore helices. Detachment of ISS contact signals the counterclockwise adjacent subunit to hydrolyze ATP.
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Comment in

  • Stepping up protein degradation.
    Maupin-Furlow JA. Maupin-Furlow JA. Proc Natl Acad Sci U S A. 2019 Jan 8;116(2):350-352. doi: 10.1073/pnas.1819949116. Epub 2018 Dec 19. Proc Natl Acad Sci U S A. 2019. PMID: 30567974 Free PMC article. No abstract available.

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