Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines

Abstract

AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use singlemolecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titin I27 domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titin I27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains.protein degradation | AAA+ proteases | directional unfolding | AAA+ motors

Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH

“…Previous studies on degradation of water-soluble proteins by ClpXP and ClpAP have shown that the pulling forces generated by ATP hydrolysis act primarily on the local structure adjacent to a degradation marker 14,44,64 . Thus, degradation strongly depends on local conformational stability as well as unfolding cooperativity of the whole structure 59 .…”

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

“…We consider both possibilities unlikely as the distribution of translocation step lengths shows little dependence on trap force (Aubin-Tam et al, 2011;Maillard et al, 2011;Sen et al, 2013;Cordova et al, 2014;Olivares et al, 2017).…”

“…Single-chain ClpX ∆N was used to ensure subunits of the pseudohexamer do not dissociate during sample preparation. This enzyme has been used previously for many biochemical and single-molecule studies (Martin et al, 2005;2008a;2008b;Aubin-Tam et al, 2011;Maillard et al, 2011;Glynn et al, 2012;Sen et al, 2013;Stinson et al, 2013;Cordova et al, 2014;Iosefson et al, 2105a;2015b;Olivares et al, 2017;Rodriguez-Aliaga et al, 2016;Amor et al, 2019;Bell et al, 2018). We purified enzymes separately and incubated ClpX ∆N (4 µM pseudohexamer), ClpP (2 µM 14-mer), and ATPγS (5 mM) for five min before vitrification.…”

“…kinetic bursts of power strokes produce fast translocation steps two, three, or four-fold larger in terms of the number of residues translocated (Aubin-Tam et al, 2011;Maillard et al, 2011;Sen et al, 2013;Cordova et al, 2014;Olivares et al, 2017). Such bursts do not occur in repeating patterns, supporting probabilistic but coordinated ATP hydrolysis within the ClpX ring.…”

“…Eliminating ATP hydrolysis in four or five subunits of single-chain pseudohexamers slows but does not prevent ClpP-mediated degradation, suggesting that ATP hydrolysis in any one of multiple subunits in the ClpX ring is sufficient to power unfolding and translocation (Martin et al, 2005). In optical-trapping experiments using single-chain ClpX and ClpP, the smallest observed translocation steps correspond to movement of five to eight amino acids of the substrate, and kinetic bursts of power strokes produce fast translocation steps two, three, or four-fold larger in terms of the number of residues translocated (Aubin-Tam et al, 2011;Maillard et al, 2011;Sen et al, 2013;Cordova et al, 2014;Olivares et al, 2017). Such bursts do not occur in repeating patterns, supporting probabilistic but coordinated ATP hydrolysis within the ClpX ring.…”