Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

Abstract

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

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 cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex

“…The ClpX∆ZBD peptide array was probed in a similar manner using purified His-TF resulting in 12 consensus motifs (motifs 1–12) in ClpX∆ZBD (Supplementary Fig. 13B, D ), which were mapped onto the cryoEM structure of ClpX∆ZBD 33 (Fig. 5a ).…”

“… A Surface representation of the 3D structure of TF (PDB:1W26) 32 and the ClpX∆ZBD hexamer (PDB:6PP5) 33 highlighting binding motifs between ClpX and TF. For the TF structure, regions that bound overlapping ClpX peptides (motifs 1–6) are shown in blue, and regions that bound overlapping TF peptides (motif 1–12) on ClpX∆ZBD (AAA+) are in red.…”