AAA+ ATPases in Protein Degradation: Structures, Functions and Mechanisms

Abstract

Adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+), the hexameric ring-shaped motor complexes located in all ATP-driven proteolytic machines, are involved in many cellular processes. Powered by cycles of ATP binding and hydrolysis, conformational changes in AAA+ ATPases can generate mechanical work that unfolds a substrate protein inside the central axial channel of ATPase ring for degradation. Three-dimensional visualizations of several AAA+ ATPase complexes in the act of substrate processing for protein degradation have been resolved at the atomic level thanks to recent technical advances in cryogenic electron microscopy (cryo-EM). Here, we summarize the resulting advances in structural and biochemical studies of AAA+ proteases in the process of proteolysis reactions, with an emphasis on cryo-EM structural analyses of the 26S proteasome, Cdc48/p97 and FtsH-like mitochondrial proteases. These studies reveal three highly conserved patterns in the structure-function relationship of AAA+ ATPase hexamers that were observed in the human 26S proteasome, thus suggesting common dynamic models of mechanochemical coupling during force generation and substrate translocation.

Keywords: 26S proteasome; AAA+ ATPase; ATP-dependent proteolysis; Cdc48/p97; mitochondrial protease; substrate translocation.

Figure 1

Domain organizations and structures of protease complex machineries of adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+). (a) Domain organizations of AAA+ proteases in different families. The length of the bar is not linearly proportional to the real length of the corresponding sequence. Each protein contains one or two AAA+ modules, each consisting of a large and small subdomain, and additional family-specific domains, which are not specifically depicted here. Protease modules reside in separate protein subunits except for FtsH and Lon. Protein subunits connected by dotted line can assemble into one complex. (b–d) Atomic models of the yeast 26S proteasome (a; PDB ID: 6FVT), the ADP-bound human p97 (b; PDB ID: 5FTK) and the substrate-bound yeast Yme1 (c; PDB ID: 6AZ0) as the representative AAA+ proteases. Orthogonal views of their proteolytic complexes/domains and hexameric ATPase rings are shown here.

Figure 2

Structure and translocation mechanism of the human 26S proteasome. (a) Cryo-EM structure of the substrate-bound human proteasome in state E_B at 3.3 Å (EMDB ID: 9218; PDB ID: 6MSE). The RPT1 density is omitted to show the substrate density inside the ATPase ring. The RPN13 density is not observed in this structure. (b) A close-up view of the quaternary interface around the isopeptide bond between substrate and ubiquitin. (c) Architecture of pore loop staircase interacting with the substrate. Aromatic residues in pore-1 loops are labelled. (d) Molecular model of RPT5 in state E_B, with ATP bound and substrate engaged. (e) Schematic of mechanical substrate translocation of proteasomal ATPases. Synchronization of nucleotide processing in three adjacent ATPases (left) causes differential vertical rigid-body rotations in each substrate-engaged ATPase that cooperatively transfer the substrate (right).

Figure 3

Structural features of the substrate-bound human 26S proteasome in different conformational states. (a) Conformational switching of the RP during the transition from state E_B (dark gray; PDB ID: 6MSE) to E_C1 (yellow; PDB ID: 6MSG), with the CP (gray) aligned. (b) Cutaway surface representations of the RP–CP interface in different states. The red dashed circles highlight the densities of the RPT C-terminal tails that are inserted into the α-pockets of the CP. (c) Schematics showing the relative locations of the pore-1 loops of six RPT subunits along the vertical axis. RPT subunits in ATP-bound (red), ADP-bound (green) and apo-like (blue) states are depicted alongside the substrate from top to bottom and subunits that are disengaged from the substrate are placed on the left side. States E_A1, E_A2 and E_C1 are omitted here, as their ATPase structures are identical to that of E_B and E_C2, respectively.

Figure 4

Schematic of coordinated ATP hydrolysis and nucleotide exchange observed in seven states in the substrate-bound human 26S proteasome [11]. Three principal modes are depicted here, with Modes 1, 2, and 3 featuring hydrolytic events in two oppositely positioned ATPases (yellow and blue), in two adjacent ATPases (orange and violet) and in one ATPase at a time (forest green), respectively. The RPT subunits with their pore-1 loops on the top and bottom of the pore-loop staircase are labeled “Top” and “Bottom”, respectively, which are consistent with Figure 3c.

Figure 5

Structure illustration of the tandem hexameric ATPases of Cdc48 processing a substrate. (a) Cryo-EM density map of the Cdc48– Ufd1-Npl4 (UN)–substrate complex in the presence of ATP and with Cdc48 carrying a Walker B mutation (EMDB ID: 0665; PDB ID: 6OA9). D1 domains of subunits E and F are both omitted for clarity. Densities of Ufd1 and N domains are too vague to be shown. (b,c) Substrate interactions with the Cdc48 D1 pore (b left) and D2 pore (c left), and corresponding nucleotide states of six ATPases (right). Only key residues in pore-1 loops are labeled here.

Figure 6

Comparison of two kinds of mitochondrial membrane-anchored AAA+ proteases. (a) Cartoon representation of i- and m-AAA proteases anchored in the mitochondrial inner membrane (IM) with opposite orientations to intermembrane space (IMS) and matrix space, respectively. (b) The pore loop spiral staircases surrounding the substrate in yeast Yme1 (PDB ID: 6AZ0; left) and human AFG3L2 (PDB ID: 6NYY; right). Aromatic residues in pore loops are all highlighted as sticks and spheres, which in pore-2 loops are colored gray.