Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase

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.

Dual Role of a Rubisco Activase in Metabolic Repair and Carboxysome Organization

“…Many AAA + proteins, including RsRca, contain an aromatic residue (Tyr114 in RsRca) in their canonical PL1 motif (aromatic-hydrophobic-glycine) that is involved in substrate peptide threading (de la Pena et al, 2018;Dong et al, 2019;Fei et al, 2020;Hanson and Whiteheart, 2005;Ripstein et al, 2020;Rizo et al, 2019;Twomey et al, 2019;Wang et al, 2020). The aromatic residues of the six PL1 loops grip and translocate the peptide substrate in a sequencepromiscuous, hydrophobic milieu.…”

“…Instead, the formation of pockets between the staggered PL1 and PL2 of adjacent Rca subunits may be the critical feature for sequence specific substrate capture. As suggested for other AAA+ proteins, peptide threading may be mediated by sequential ATP hydrolysis around the hexamer ring (de la Pena et al, 2018;Dong et al, 2019;Fei et al, 2020;Ripstein et al, 2020;Rizo et al, 2019;Twomey et al, 2019;Wang et al, 2020).…”

“…The AAA+ chaperones are typically substrate-promiscuous and act by threading flexible terminal sequences or loop segments of their target proteins into the hexamer pore (Olivares et al, 2016;Puchades et al, 2020). Threading is mediated by pore-loops ( Figure 1A) that face the central pore and engage the substrate peptide, exerting a pulling force (Avellaneda et al, 2020;de la Pena et al, 2018;Dong et al, 2019;Fei et al, 2020;Ripstein et al, 2020;Rizo et al, 2019;Twomey et al, 2019).…”

Functional analysis of proposed substrate-binding residues of Hsp104

“…These findings suggest that this third noncanonical pore loop centered on Y650 might play an important role in substrate binding and translocation. Furthermore, in a cryo-EM structure of ClpB, the bacterial homolog of Hsp104, a similar third loop centered around H641 projects into the channel and appears to interact with the substrate polypeptide [27]. In a luciferase reactivation assay ClpB H641A showed a loss of~50% activity relative to ClpBWT [27].…”

“…Furthermore, in a cryo-EM structure of ClpB, the bacterial homolog of Hsp104, a similar third loop centered around H641 projects into the channel and appears to interact with the substrate polypeptide [27]. In a luciferase reactivation assay ClpB H641A showed a loss of~50% activity relative to ClpBWT [27]. However, there are many key differences between Hsp104 and ClpB, and importantly ClpB has only limited activity against amyloid structures [12].…”