Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

Abstract

AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.

Keywords: AAA+; Hsp100; chaperone; cryo-EM; protein disaggregation; protein unfolding.

Graphical abstract

Figure 1

ClpB Activation Triggers a Sequential Mode of ATP Hydrolysis (A) ClpB domain organization and monomer structure. The identity and position of mutated residues are indicated. (B) ATPase activities of ClpB wild-type (WT) and ClpB-K476C were determined in the absence and presence of 10 μM casein (± substrate). SDs are indicated; for some points, error bars are shorter than the height of the symbol and are not depicted. (C) MDH disaggregation activities of ClpB-WT and ClpB-K476C in the absence and presence of Hsp70. (D) ATPase activity of ClpB-WT and ClpB-K476C in absence and presence of casein (± substrate) as a function of ATP concentration. (E) v_max of ATPase activities, derived Hill coefficient (h), and ATP concentrations at half-maximal ATPase activity (K_0.5) for WT, pore 1 (Y251A), and pore 2 (Y653A) loop mutants of ClpB-WT and ClpB-K476C. (F and G) ATPase activities of ClpB-K476C/ClpB-K476C/E279A/E678A (F) and MDH disaggregation of ClpB-WT/ClpB-E279A/E678A (G) mixes were determined (red, blue). They are compared with curves calculated from a model (black to gray) that assumes that a mixed hexamer only displays ATPase or disaggregation activity if it contains the number of wild-type subunits indicated. Mixing ratios are indicated as number of E279A/E678A mutant subunits.

Figure 2

Overview of Substrate-Bound ClpB-DWB-K476C (A) Left, top view, and middle and right, side views of the cryo-EM density map of the most populated conformation of casein-bound ClpB-DWB-K476C (KC-2). The six protomers form a closed ring with a helical arrangement of two stacked AAA tiers and a seam between subunits A and F. The flexible N-terminal domains, located above the AAA1 tier, are not visible at high contour level. M-domains are partly visible for protomers C–E. (B) Views of the cryo-EM maps of the three states of substrate-bound ClpB-DWB-K476C. Densities of protomers A and B are removed to show conformational changes in protomers AAA1E and AAA2F, highlighted by orange and red arrows, respectively. Orange and red hexagons show the position of moving AAA1E and AAA2F pore loops.

Figure 3

Pore Loop Movements and Arginine Finger Contacts in the Three States of Substrate-Bound ClpB-DWB-K476C Suggest a Sequential Mechanism of ATP Hydrolysis and Substrate Threading (A) Pore loop interactions with the substrate in AAA1 (top panels) and AAA2 (bottom panels) rings. The pore loop AAA1E (orange) engages the substrate in KC-2, while the pore loop AAA2F (red) dissociates. AAA2F moves from the bottom to the top of the staircase of pore loops in KC-3. (B) Arginine finger engagements in the AAA1 and AAA2 ring. All protomers were aligned to the large lobe of AAA1 or AAA2 domain of protomer C to compare engagement of the arginine fingers with neighboring subunits. Arginine fingers of AAA1B–C and AAA2B–D are shown as gray ribbons and interact with the γ-phosphate of ATP bound at the active site of a neighboring subunit in all three states. Activity states of AAA2 protomers are indicated by green (active) and red (inactive) arrows. (C) Nucleotide densities for AAA2A, AAA2B, and AAA2F protomers and assigned nucleotide state.

Figure 4

Activation and Inactivation of Subunits in the AAA2 Ring Are Directly Coupled (A) Views of the AAA2 domains of protomers A, B, E, and F for states KC-2 and KC-3 are shown. The small lobe of AAA2B is omitted for clarity. In state KC-2 (left), the arginine finger of AAA2A (highlighted by yellow oval) contacts the γ-phosphate of ATP bound at neighboring AAA2B. In the post-hydrolysis state KC-3 (right), detachment of this arginine finger allows rotation of AAA2A by 14° to move away from AAA2B while remaining bound to the substrate. This rotation is transmitted to AAA2F, causing its repositioning to the top of the spiral track of AAA2 pore loops and its activation by receiving an arginine finger from AAA2E. (B) The track of Cα atoms when morphing from KC-2 to KC-3 illustrates the amplitude of movements of AAA2A and AAA2F at the seam of the AAA2 ring. Blue and red arrows highlight the rotations of AAA2A and AAA2F subunits, respectively. (C) Activation of AAA1E is a prerequisite for AAA2F rotation. Views of AAA1E, AAA2F, and casein substrate in states KC-1, KC-2, and KC-3 are shown.

Figure 5

Docked M-Domains Repress the Activity of ClpB-WT and Reduce the Range of AAA Domain Movements (A) Heterogeneity of M-domain conformations. Top and side views of the cryo-EM density maps for the two main M-domain conformations of the ClpB-DWB-K476C:casein complex and for the ClpB-DWB:casein complex. Detached M-domains are indicated by green arrows and docked M-domains by red arrows. (B) Docking states of M-domains: atomic models showing the predominant conformation of M-domains enclosing the AAA1 tier in ClpB-K476C (left) and ClpB-WT (right) states. In ClpB-WT, M-domains are docked in a horizontal conformation that is stabilized by head-to-tail interactions between motif 1 and motif 2 of neighboring M-domains. In ClpB-K476C, M-domains adopt a tilted conformation with motif1 contacting the AAA1 domain of the adjacent protomer. Head-to-tail interactions are broken, rendering M-domain motif 2 invisible in the cryo-EM maps. Here, full-length M-domains are shown, docked in the density of motif1, to emphasize the differences in M-domain docking states between ClpB-WT and ClpB-K476C.

Figure 6

Overview of Substrate-Bound ClpB-DWB, Pore Loop Movements and Arginine Finger Contacts (A) Views of the cryo-EM maps of the three states of substrate-bound ClpB-DWB. Densities of protomers A and B were removed to show conformational changes in protomers AAA1E and AAA2F, highlighted by orange and red arrows, respectively. Orange/red hexagons show the position of moving pore loops. (B) Interactions of ClpB-WT pore loops of the AAA1 (upper panel) and AAA2 (lower panel) rings with the substrate. The pore loop AAA2F (red) dissociates from the substrate in WT-2A. The pore loop AAA1E (orange) binds substrate in WT-2B. (C) Activity states of ClpB-WT AAA1 (upper panels) AAA2 (lower panels) domains. All protomers were aligned to the large AAA1 (AAA2) domain of protomer C to compare engagement of the arginine fingers with neighboring subunits. Arginine fingers of AAA1A-C and AAA2B-D are shown as grey ribbons and interact with the γ-phosphate of ATP bound at the active site of a neighboring subunit in all three states. Activity states of AAA1/2 protomers are indicated by green (active) and red (inactive) arrows. (D) Comparison of ClpB-WT and ClpB-K476C pore loop positions of AAA1E and AAA2F for states WT-1, WT-2A to WT-2B (pale to bright colors) and for states KC-1, KC-2 to KC-3 (pale to bright colors).

Figure 7

Coupled Sequential ATP Hydrolysis and Polypeptide Translocation upon ClpB Activation (A and B) Schematic representation of AAA1 and AAA2 rings (A) and side views of A, E, and F subunits bound to the substrate (B). Nucleotide states are shown in pink, pale pink, and white for ATP, ADP, and apo states, respectively. Engaged and detached arginine fingers are shown as green triangles and red crosses, respectively. Green, yellow, and red segments indicate activity states of AAA domains (active, intermediate, and inactive states, respectively). The gradient arrow shows pore loop position on the substrate from lowest in gray to highest in black. In this model, the AAA2 ring drives substrate threading, and the AAA1 ring follows conformational changes initiated in the AAA2 ring. Binding of ATP to apo AAA2F leads to its dissociation from the substrate and ADP release in AAA2A (state 2). ATP hydrolysis in AAA2B, triggered by the presence of apo AAA2A, allows detachment of the AAA2A arginine finger and repositioning of AAA2F to the top position (state 3). In the subsequent step (state 1 + 1) AAA2F contacts the substrate while AAA1A dissociates, resulting in the same conformation as state 1 but shifted by one protomer counterclockwise. This propels the substrate in discrete steps, ultimately causing the extraction of a polypeptide from a protein aggregate.