High-resolution cryo-EM structure of the proteasome in complex with ADP-AlFx

Abstract

The 26S proteasome is an ATP-dependent dynamic 2.5 MDa protease that regulates numerous essential cellular functions through degradation of ubiquitinated substrates. Here we present a near-atomic-resolution cryo-EM map of the S. cerevisiae 26S proteasome in complex with ADP-AlFx. Our biochemical and structural data reveal that the proteasome-ADP-AlFx is in an activated state, displaying a distinct conformational configuration especially in the AAA-ATPase motor region. Noteworthy, this map demonstrates an asymmetric nucleotide binding pattern with four consecutive AAA-ATPase subunits bound with nucleotide. The remaining two subunits, Rpt2 and Rpt6, with empty or only partially occupied nucleotide pocket exhibit pronounced conformational changes in the AAA-ATPase ring, which may represent a collective result of allosteric cooperativity of all the AAA-ATPase subunits responding to ATP hydrolysis. This collective motion of Rpt2 and Rpt6 results in an elevation of their pore loops, which could play an important role in substrate processing of proteasome. Our data also imply that the nucleotide occupancy pattern could be related to the activation status of the complex. Moreover, the HbYX tail insertion may not be sufficient to maintain the gate opening of 20S core particle. Our results provide new insights into the mechanisms of nucleotide-driven allosteric cooperativity of the complex and of the substrate processing by the proteasome.

Figure 1

Proteasome proteolytic activity and cryo-EM structure of the single-capped 26S proteasome-ADP-AlFx. (A) In vitro proteolytic activity of proteasome toward a fluorogenic peptide substrate Suc-LLVY-AMC in different nucleotide states. RFU, relative fluorescence units. (B) Relative hydrolysis ability of proteasome toward the fluorogenic substrate was analyzed for the same samples as in A. Standard deviations were calculated from three independent experiments. (C) The cryo-EM map of proteasome-ADP-AlFx in different views (dodger blue). (D) The corresponding pseudo-atomic model fitted into the proteasome-ADP-AlFx map, with the models for different 19S RP subunits in color and that for 20S CP in dodger blue. Different subunits in 19S RP are labeled. (E) Representative high-resolution α helix and β strand features of the 20S β subunits (β1 and β2) in the proteasome-ADP-AlFx map. The β strand separation can be well resolved. The selected residues with bulky side chain are labeled.

Figure 2

A new AAA-ATPase ring conformational configuration in proteasome-ADP-AlFx map. The AAA-ATPase ring structure of proteasome-ADP-AlFx elucidates two obvious gaps beside Rpt2 subunit (indicated by two red stars). The map is rendered as transparent surface and the model is in color. The interaction interface (in Ų) between the neighboring AAA domains is labeled. The color scheme for the map and model of individual AAA-ATPase subunit is followed throughout. The map here is low-pass filtered to 5.5-Šresolution (local resolution in this region) for better visualization.

Figure 3

A distinct configuration of pore loops in the AAA-ATPase ring of proteasome-ADP-AlFx. (A) Cutaway side view of the AAA-ATPase ring of proteasome-ADP-AlFx, with the upper location of each of the pore loops denoted by two red spheres. A noticeable “lockwasher-like” configuration of the pore loops can be observed (indicated by a black curve with an arrow head), with Rpt5 and Rpt1 forming the beginning and the end of the “lockwasher”, respectively. (B) The AAA domain of individual AAA-ATPase subunit shown in the same orientation. The pore loop is shown as thick red loop and the height of the pore loops is denoted by the dashed black line. Same low-pass filtered map as in Figure 2 is used here. (C) The overlaid AAA domains from proteasome-ADP-AlFx (colored) and proteasome-ATPγS (S3, PDB ID: 4CR4, transparent salmon), with the vertical differences between their positions in the two structures indicated. (D) The overlaid AAA domains from proteasome-ADP-AlFx (colored) and substrate-engaged proteasome (grey), with the vertical differences between their positions indicated. The model of the substrate-engaged proteasome structure was generated from PAN (PDB ID: 3H4M).

Figure 4

Distinct nucleotide occupancy statuses between proteasome-ADP-AlFx and resting state proteasome. (A) Nucleotide occupancy status in the nucleotide-binding pocket of individual AAA-ATPase subunit in the proteasome-ADP-AlFx map. We used ADP to locate the approximate position of potential nucleotide. The map reveals relative strong density in the nucleotide-binding pockets of four consecutive subunits (Rpt3-Rpt4-Rpt5-Rpt1), no obvious extra density in the Rpt2 pocket, and rather weak density for potential nucleotide in the Rpt6 pocket. Same low-pass filtered map as in Figure 2 is used here. (B) Our yeast resting state structure reveals that the nucleotide-binding pockets of all the six AAA-ATPase subunits are occupied. In both maps, due to resolution limitation in 19S RP region, we are not able to distinguish different nucleotide forms (ADP-AlFx, ADP, or ATP) in the individual pocket.

Figure 5

Insertion of the AAA-ATPase subunit C-terminal HbYX motif into 20S is not sufficient to trigger 20S gate opening. (A) End-on view of 20S with the inserted C-terminal tails of Rpt2, Rpt3, and Rpt5 (red pieces of density) in the proteasome-ADP-AlFx (dodger blue) and resting state (grey) maps. The gate region is indicated by a dash line square. (B) Superimposed models showing only the gate region, with that of resting state in grey, that of proteasome-ADP-AlFx state in dodger blue, and that of the gate closed eukaryotic 20S crystal structure (PDB ID: 1RYP) in sandy brown.