Mechanochemical basis of protein degradation by a double-ring AAA+ machine

Abstract

Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli mechanically degrades proteins in complex with the ClpP peptidase. We demonstrate that ClpA unfolds some protein substrates substantially faster than the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

“…This type of helical arrangement is often observed for other AAA+ proteins, such as the yeast replication factor C (61), the bacterial DnaA AAA+ domain (62), the bacterial IstB domain (63), and the eukaryotic MCM E1 helicase (64), implying a potential evolutionary conservation in the architecture of these proteins despite varying structural features and distinct biological functions. It has been proposed that VAT functions via a substrate threading mechanism (18) similar to the mechanism used by other AAA+ unfoldases, such as ClpX and ClpA (65,66), whereby protein substrates are unfolded and threaded through the central pore of the unfoldase and subsequently passed into the degradation chamber of the 20S proteasome CP. The mechanical force for this process is generated from repeated cycles of ATP binding and hydrolysis.…”

“…The mechanical force for this process is generated from repeated cycles of ATP binding and hydrolysis. Substrates are pulled through the narrow channel of the unfoldase via attachment to pore loops located at the entrance and in the interior of the channel (65,(67)(68)(69)(70), which also play a role in substrate recognition in some AAA+ machines (5,(71)(72)(73). In the case of VAT, mutations of pore loop residues Y264 in D1 and W541/V542 in D2 cause severe defects on substrate unfolding and translocation (18).…”

Two Isoforms of Clp Peptidase in Pseudomonas aeruginosa Control Distinct Aspects of Cellular Physiology

“…Peptidase assays used 10% dimethyl sulfoxide (DMSO), 2.0 mM N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin (Suc-LY-AMC) (Bachem), and 0.4 M ClpP tetradecamer (ClpP 14 ), and degradation was monitored by fluorescence with excitation at 345 nm and emission at 440 nm. Degradation of GFP with a C-terminal SsrA degron tag was monitored as previously described using 0.2 M ClpP 14 and 0.1 M ClpX 6 and an ATP regeneration system (44). ATP hydrolysis rates of ClpX were monitored with 0.1 M ClpX hexamer (ClpX 6 ) and 0.2 M ClpP 14 , as previously described (27).…”

The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

“…As the force is applied across the protein domains the activation barrier for unfolding is lowered, increasing the probability that the protein will unfold. Studies have helped identify the role of mechanical unfolding forces in protein degradation [58][59][60]. They have uncovered details of force generation in motor proteins [61,62] and the importance of plasticity of hydrogen bond networks in regulating the mechanochemistry of cell adhesion complexes [63], cell signalling (mechanosensors) [64], force generation [61,62] and cell signalling [65,66].…”