Structural basis for intersubunit signaling in a protein disaggregating machine

Abstract

ClpB is a ring-forming, ATP-dependent protein disaggregase that cooperates with the cognate Hsp70 system to recover functional protein from aggregates. How ClpB harnesses the energy of ATP binding and hydrolysis to facilitate the mechanical unfolding of previously aggregated, stress-damaged proteins remains unclear. Here, we present crystal structures of the ClpB D2 domain in the nucleotide-bound and -free states, and the fitted cryoEM structure of the D2 hexamer ring, which provide a structural understanding of the ATP power stroke that drives protein translocation through the ClpB hexamer. We demonstrate that the conformation of the substrate-translocating pore loop is coupled to the nucleotide state of the cis subunit, which is transmitted to the neighboring subunit via a conserved but structurally distinct intersubunit-signaling pathway common to diverse AAA+ machines. Furthermore, we found that an engineered, disulfide cross-linked ClpB hexamer is fully functional biochemically, suggesting that ClpB deoligomerization is not required for protein disaggregation.

Fig. 1.

Crystal structure the intact ClpB D2 domain. (A) Ribbon diagram of the D2 domain (gold) with the pore loop colored in magenta (PDB entry code: 4FCV_A). ADP is shown as stick model. The orientation of the D2 domain in the hexamer and relative to the sixfold axis (arrow) is indicated. (Inset, Left) Enlarged view of the nucleotide-binding pocket. The simulated annealed omit map was calculated with ADP omitted and is contoured at 1.5σ. (Inset, Right) Enlarged view of the pore loop, with the substrate-interacting Tyr643 highlighted in cyan. The 2F_o - F_c map is contoured at 1.0σ. (B) Top-down view of the fitted cryoEM structure of the D2-ADP (Left) and apo D2 hexamer (Right). The cryoEM maps are shown as semitransparent surfaces. Neighboring subunits are colored differently for clarity.

Fig. 2.

Functional analysis of a disulfide cross-linked ClpB hexamer. (A) Position of engineered disulfide pairs, which were introduced into full-length ClpB. Only the D2 ring is shown. Mutation sites are depicted as spheres, and cysteine pairs are colored in different hues: ClpB_R576C/A821C in pink and magenta, ClpB_L581C/R776C in light and dark green, and ClpB_N746C/R810C in light and dark brown. Neighboring subunits are shown in different gray shades for clarity. Bound nucleotide is depicted as stick model. (B) ClpB_R576C/A821C, ClpB_L581C/R776C, and ClpB_N746C/R810C form high molecular weight, cross-linked oligomers in the presence of ATP after 10 min of cross-linking reaction. In contrast, ClpB, ClpB_R576C, and ClpB_L581C/R726C that feature a mismatched cysteine pair do not. An asterisk marks the product when performing the cross-linking reaction on ice. (C) Time course of catalyzed cross-linking reaction of ClpB_R576C/A821C in the presence of ATP. M, marker; C, disulfide cross-linked ClpB_R576C/A821C hexamer after 20 min; G, glutaraldehyde crosslinked ClpB_E271A/E668A hexamer. (D) Size-exclusion chromatograms of wild-type (wt) ClpB and crosslinked (xl) ClpB_R576C/A821C. (E–G) ATPase (E) and coupled chaperone activities (F, G) of cross-linked ClpB_R576C/A821C relative to wild-type (wt) ClpB, non-crosslinked ClpB_R576C/A821C, and ClpB_R576C. Error bars represent standard deviations of three independent experiments. (H) Coupled chaperone activities of cross-linked ClpB_R576C/A821C and cysteine-free ClpB variants featuring the L460A or L396A mutation, or both. Error bars represent standard deviations of three independent experiments.

Fig. 3.

Structural determinants coordinating ATP binding and hydrolysis in the D2 ring. (A) Arg747 reaches out from one subunit (gold) into the ATP-binding site of its neighbor (cyan). ADP is shown as stick model. (B) The R747A mutation abolishes the residual ATPase activity of ClpB_E271A, whereas the R741A mutation did not, indicating that Arg747 is the Arg-finger. Error bars represent standard deviations of three independent experiments. (C) Stereo diagram of the interaction between the pore loop (magenta) and the β-hairpin (stick model) in the D2-ADP structure. Asp685 is highlighted in green and the β-hairpin loop deletion in blue. The orientation of the pore loop relative to the hexamer and sixfold axis (arrow) is indicated. (D–F) Basal and casein-stimulated ATPase (D) and coupled chaperone activities (E, F) of ClpB, ClpB_Y643A, and ClpB variants in which structural elements of the ISS motif (ClpB_D685A) or β-hairpin (ClpB_H693A, ClpB_Δ691–695, ClpB_{Δ690–696/GGG}) were mutated.

Fig. 4.

Model of the intersubunit-signaling cascade in the ClpB D2 ring. Proposed model of the ISS pathway, which regulates the ATP power stroke that drives substrate translocation through the D2 ring. The D2 pore loop is colored magenta and the ISS network orange/green/blue.