Protein knots provide mechano-resilience to an AAA+ protease-mediated proteolysis with profound ATP energy expenses

In this study, we directly monitored the ClpXP-mediated degradation of UCH variants by electrophoresis and quantitative imaging analyses. We demonstrated that untying of the 52 knot in UCHL1 via N-terminal truncation (UCHL1Δ11) significantly reduces its mechanostability. We further quantified the ATP expenditures of degrading different UCH variants by ClpXP. The unknotted UCHL1Δ11 underwent accelerated ClpXP-dependent proteolysis, with a 30-fold reduction in ATP consumption compared to the knotted wild type. Unlike all other known ClpXP substrates, UCHL5, which is the most resilient substrate known to date, significantly slowed down the ATP turnover rate by ClpXP. Furthermore, UCHL5 required 1000-fold more ATP to be fully degraded by ClpXP compared to GFP. Our results underscored how the complex, knotted folding topology in UCHs may interfere with the mechano-unfolding processes of the AAA+ unfoldase, ClpX.Graphical abstract
Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - Category: Biochemistry Source Type: research