Abstract
Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.
Highlights
Bacterial ClpB and yeast Heat-shock protein (Hsp)[104] are homologous Hsp[100] protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates
Alignment of the protomers reveals substantial NBD1–NBD2 conformational changes occur between these protomers, which primarily involve rotations of the NBD1–NBD2 connecting residues (545–555) (Supplementary Movie 2) and appear to coincide with substrate release and ATP hydrolysis (Fig. 7d). From these data we propose a model in which the NBD1–NBD2 connecting residues function as a nucleotidedriven swinging arm or spring that alters the positions of NBD1 a b
For the Hsp[100] disaggregases, key questions have remained about the specific roles of the two AAA + domains, NBD1 and NBD2, and how conformational changes and ATP hydrolysis might be tuned in the different homologs to drive polypeptide translocation
Summary
Bacterial ClpB and yeast Hsp[104] are homologous Hsp[100] protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. 1234567890():,; Heat-shock protein (Hsp) 100 protein complexes are critical cell stress responders that solubilize and unfold toxic protein aggregates and amyloids, thereby enhancing thermal and chemical tolerances[1,2] They are members of the conserved family of AAA+ molecular machines that form dynamic hexameric-ring structures and undergo ATP hydrolysisdriven translocation of polypeptide substrates through a central channel[3,4,5,6]. Recent cryo-EM structures of substrate-bound Hsp1046 and ClpB5,37 identify an asymmetric spiral architecture of the hexamer that stabilizes polypeptide substrate in the channel via poreloop contacts from five protomers, while a sixth protomer at the interface of the spiral is unbound This architecture is similar to other recent structures of single and double-ring AAA+ complexes[38,39,40,41], supporting a conserved substrate interaction mechanism. Considering the DWB likely uncouples conformational changes from the hydrolysis cycling that is required for disaggregation, how ATP hydrolysis synchronizes distinct hexamer conformations to drive translocation remains a key question
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