Stairway to translocation: AAA+ motor structures reveal the mechanisms of ATP-dependent substrate translocation

Abstract

Translocases of the AAA+ (ATPases Associated with various cellular Activities) family are powerful molecular machines that use the mechano-chemical coupling of ATP hydrolysis and conformational changes to thread DNA or protein substrates through their central channel for many important biological processes. These motors comprise hexameric rings of ATPase subunits, in which highly conserved nucleotide-binding domains form active-site pockets near the subunit interfaces and aromatic pore-loop residues extend into the central channel for substrate binding and mechanical pulling. Over the past 2 years, 41 cryo-EM structures have been solved for substrate-bound AAA+ translocases that revealed spiral-staircase arrangements of pore-loop residues surrounding substrate polypeptides and indicating a conserved hand-over-hand mechanism for translocation. The subunits' vertical positions within the spiral arrangements appear to be correlated with their nucleotide states, progressing from ATP-bound at the top to ADP or apo states at the bottom. Studies describing multiple conformations for a particular motor illustrate the potential coupling between ATP-hydrolysis steps and subunit movements to propel the substrate. Experiments with double-ring, Type II AAA+ motors revealed an offset of hydrolysis steps between the two ATPase domains of individual subunits, and the upper ATPase domains lacking aromatic pore loops frequently form planar rings. This review summarizes the critical advances provided by recent studies to our structural and functional understanding of hexameric AAA+ translocases, as well as the important outstanding questions regarding the underlying mechanisms for coordinated ATP-hydrolysis and mechano-chemical coupling.

Keywords: AAA+ ATPases; cryo-EM; spiral staircase; translocation.

Figure 1

Conserved residues and motifs in AAA+ ATPase motors. (a) Nucleotide‐binding pocket at the interface between the ATPase subunits Rpt1 (green) and Rpt2 (yellow) of the 26S proteasome (PDB:5L4G).74 Highlighted are the intra‐subunit Walker‐A [K/T] (magenta), Walker‐B [E] (purple), and Sensor‐1 [N] (lime green) motifs, as well as the Arginine Finger [R/R] (pink) provided by the clockwise‐next subunit. ATP is shown in cyan and orange, and Mg²⁺ as a lime‐green sphere. (b) A cryo‐EM density representative “Top” view of the proteasomal AAA+ motor domains (EMDB:4002). The large and small AAA+ subdomains are shown in dark and light green, respectively, for Rpt1, and in dark and light yellow for the neighboring Rpt2. ATP in the binding pocket near the subdomain and subunit interfaces is colored red. Pore‐1 loops are colored orange and the central channel is circled. The remaining Rpt protomers in the hexamer are shown in shades of gray

Figure 2

Spiral‐staircase arrangement of pore‐1‐loop residues in contact with substrate. (a) Atomic model (PDB:6EF2) and EM density (EMDB 9440) for the spiral staircase of pore‐1‐loop residues in the 5T state of the 26S proteasome. Shown are the substrate in magenta as well as the conserved Tyr and flanking Lys residues for each protomer numbered by the position in the “spiral staircase,” with Rpt1 in green, Rpt2 in yellow, Rpt6 in blue, Rpt3 in orange, Rpt4 in red, and Rpt5 in cyan. (b) The pore‐1‐loop Tyr's and the substrate polypeptide from the 5T model of the 26S proteasome (56) are displayed in gray and overlaid with 1‐Å centroids indicating the Cα positions for the aromatic “paddle” residues in the pore‐1 loops of substrate‐bound YME1 (6AZ0), AFG3L2 (6NYY), VPS4 (5UIE, 6BMF, 6AP1), the 26S proteasome (1D*‐state (6EF0), 5D‐state (6EF1), 5T‐state (6EF2), 4D‐state (6EF3), E_D1‐state (6MSJ), E_D2‐state (6MSK)), D2 domains of VAT/p97 (5VCA), D2 domains of NSF (6MDO, 6MDP), D2 domains of Rix7 (6MAT), D2 domains of Hsp104 in the closed (5VJH) and extended states (5VYA), D2 domains of ClpB D2 (ATPγS‐bound double‐WB mutant (5OFO), KC‐1 state (6qs6), KC‐2A state (6qs7), KC‐2B state (6qs8), KC‐3 state (6qs4), WT‐1 (6rn2), WT‐2A (6rn3), WT‐2B (6rn4), conformer1 (6DJU), conformer2 (6DJV), Pre (6OAX), Post (6OAY)), TRIP13 (6F0X), D2 domains of Hsp101 (6E10), and Spastin (6P07). The Cα's for the aromatic pore‐1‐loop residue of protomers 1–5 in each motor were aligned to the proteasomal 5T state as a reference model (6EF2), using Matchmaker in Chimera.75 RMSD values for this alignment ranged from 0.403–1.526 Å. Centroids for the substrate‐disengaged subunit were omitted, as they have the largest variance in position. The two green centroids that do not overlay are from AFG3L2 (6NYY) and 1D* (6EF0), which both have two substrate‐disengaged subunits

Figure 3

Sequence alignment for the pore‐1‐loop residues (boxed) and flanking amino acids in the AAA+ motor domains of protein translocases, including both the D1 and D2 domains for the double‐ring Type II AAA + 's. Abbreviations are as follows: Homo sapiens (HS), Saccharomyces cerevisiae (SC), Drosophila melanogaster (DM), Thermoplasma acidophilum (TA), Cricetulus griseus (CG), Mycobacterium tuberculosis (TB), Escherichia coli (EC), and Plasmodium malariae (PM). Alignments were done in MUSCLE76 and illustrated with AlignmentViewer 77

Figure 4

The nucleotide state of a AAA+ subunit depends on its position within the spiral staircase. (a) Cartoon representation of the spiral staircase arrangement with five substrate‐engaged and one dis‐engaged subunit, depicting their nucleotide states. ATP‐bound protomers are colored green, with a lighter green in position 4 indicating some accounts of ADP and potential hydrolysis in this subunit. The substrate‐disengaged “seam” subunit is colored red to represent an ADP‐bound or apo state, while the “Bottom” subunit is shown in orange to indicate a mostly ADP‐bound state, with some accounts of ATP in this position. (b) EM density for the “KC‐2” state of ClpB (EMDB:4625, complete hexamer shown in the inset with D1 domains in light blue and D2 domains in dark blue) reveals the slanted conformation observed in double ring AAA+ motors. The D1 and D2 domains are shown in light and dark blue for protomer 3, and in light and dark orange for protomer 4, highlighting how the D1 domain of a subunit overlaps with the D2 domain of the counterclockwise‐next neighbor