AAA-ATPases in Protein Degradation

Abstract

Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

Keywords: AAA; ATPase; proteasome; protein folding; proteolysis.

Figure 1

Three functional states of the eukaryotic proteasome. Conformational states of the eukaryotic proteasome, S1 (Left), S2 (Middle), and S3 (Right). The alpha ring of the catalytic core particle (CP) is colored in purple with full views of the regulatory particle (RP) for S1/S2 and longitudinal cross section for S3. The central channel through the CP and ATPase ring is indicated with yellow and white dashed lines, respectively. In the RP, the AAA-ATPase ring along with the N-terminal coiled-coils is colored in cyan. The non-ATPase RP subunits are colored in white except for Rpn1 (brown), Rpn2 (green), Rpn10 (orange), Rpn11 (yellow), and Rpn13 (magenta). In S1, a poly-ubiquitylated substrate (in red labeled with “S” attached to the tetra-ubiquitin chain in blue labeled with “Ub”) is recognized by the ubiquitin receptor Rpn13. Subsequently, the poly-ubiquitin chain is anchored to the ubiquitin receptor Rpn10 leading to substrate placement near the N-ring. In S2, the isopeptide bond between the substrate and poly-ubiquitin chain is cleaved by Rpn11 and the unfolding of the substrate is initiated. In S3, the unfolded substrate is translocated through the central pore of the AAA-ATPase ring into the central channel of the CP for degradation. The central pores of the AAA-ATPase O-ring and the CP are not aligned in S1 and S2 but are in S3. A 25° rotation of S1 to S2 facilitates the substrate placement into the N-ring and activates Rpn11. The figure was prepared using the PDB IDs: 4CR2, 4CR3, 4CR4, 1UBQ, 2ZNV, 2Z59, and 1UZX through PyMOL (Ver. 1.8.0.2) molecular graphics software (Schrodinger, LLC, New York).

Figure 2

Domain organization of AAA-ATPases. (A) Magnified view of the monomer (left) and overall view of the oligomer (right) of Mpa containing two OB rings, OB1 and OB2, along with the N-terminal coiled-coils (blue). Magnified views of monomers bound to nucleotides highlighted by spheres of (B) ClpA with small and large domains SD1, SD2, LD1, and LD2 bound to ADP at the SD1/LD1 and SD2/LD2 interfaces; of (C) Valosin-containing protein-like ATPase (VAT) with nucleotide binding domains NBD1 and NBD2 bound to ATP; of (D) HslU with N-terminal (N), large (LD), and small (SD) domains and ATP; of (E) ClpX with N-terminal (N), large (LD), and small (SD) domains with ADP; of (F) p97/VCP/Cdc48 with N-terminal (N) and domain-1 (D1) and -2 (D2) bound to ATPγS; of (G) proteasome-activating nucleotidase (PAN) with N-domains 1 (from Gcn4) and 2 and large (LD) and small (SD) domains. Again, ATP is bound at the SD/LD interface. This figure was prepared based on the availability of structures in the protein data bank using the PDB IDs: 3M9D, 1KSF, 5VC7, 1DO0, 3HWS, 5C18, 2WG5, and 2WFW through PyMOL (Ver. 1.8.0.2) molecular graphics software (Schrodinger, LLC, New York).

Figure 3

Active site organization of AAA-ATPase rings. (A) Bottom view of the proteasomal AAA-ATPase rings from yeast (upper panel) and human (lower panel). The Walker domain A is highlighted by red spheres and B by blue spheres. Magnified views of the Walker domains are shown for human AAA-ATPase bound to either ATP (B) or ADP (C) in two orientations. (D) Dynamics of Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT) are visualized by conformational switches between the stacked and spiral (split-) ring versions. Side and top views of the AAA-ATPase subunit colored in red show movements out of the plane upon ATP hydrolysis aiding substrate translocation into the proteasome through its central pore. The split ring form (bottom left) undergoes a conformational change back into the stacked ring (top left), when ADP dissociates from the subunit and ATP binds back to allow the next round of hydrolysis. This figure was prepared using the PDB IDs: 4CR2, 5L4G, 5G4G, and 5G4F through PyMOL (Ver. 1.8.0.2) molecular graphics software (Schrodinger, LLC, New York).

Figure 4

The AAA+ ATPase ring of the yeast 26S proteasome. (A) Structure of an assembled yeast proteasome showing half of the catalytic core particle (CP) attached to the regulatory particle (RP). The AAA-ATPase ring is highlighted in colors. (B) A magnified view of the AAA-ATPase ring containing the subunits Rpt1 to Rpt6. The N-terminal coiled-coils are formed by Rpt1 and 2, Rpt4 and 5, and Rpt3 and 6. (C) The interface between the ATPase ring and the CP is shown with the HbYX motif at the C-terminus of Rpt3 (highlighted as yellow spheres) digging into the alpha subunit binding pocket of the CP. Residues of CP alpha subunits that line the binding pocket of the HbYX motif are highlighted in magenta. This figure was prepared using the PDB ID: 4CR2 through PyMOL (Ver. 1.8.0.2) molecular graphics software (Schrodinger, LLC, New York).

Figure 5

The AAA-ATPase ring of the human proteasome. (A) The AAA-ATPase is located on the alpha ring of the CP. The catalytic beta-subunits are colored in red, the alpha-subunits in blue. A magnified view of the AAA-ATPase ring is shown on the right. Coiled-coils of N-terminal regions reach out to other RP subunits. (B) The AAA-ATPase subunit colored in cyan is bound to ADP (red ellipse), while the other five AAA-ATPase subunits are bound to ATP (red box). (C) Rpn3 acts as sensor to induce conformational changes in the RP upon substrate docking into the ATPase ring (shown as a surface diagram). The C-terminus of Rpn3 colored in red is close to the pore of the N-ring (white line) and the O-ring (yellow line). This figure was prepared using the PDB ID: 5L4G through PyMOL (Ver. 1.8.0.2) molecular graphics software (Schrodinger, LLC, New York).