Topologically knotted deubiquitinases exhibit unprecedented mechanostability to withstand the proteolysis by an AAA+ protease

Abstract

More than one thousand knotted protein structures have been identified so far, but the functional roles of these knots remain elusive. It has been postulated that backbone entanglement may provide additional mechanostability. Here, we employed a bacterial proteasome, ClpXP, to mechanically unfold 52-knotted human ubiquitin C-terminal hydrolase (UCH) paralogs from their C-termini, followed by processive translocation into the proteolytic chamber for degradation. Our results revealed unprecedentedly slow kinetics of ClpXP-mediated proteolysis for the proteasome-associated UCHL5: ten thousand times slower than that of a green fluorescence protein (GFP), which has a comparable size to the UCH domain but much higher chemical and thermal stabilities. The ClpXP-dependent mechanostability positively correlates with the intrinsic unfolding rates of the substrates, spanning over several orders of magnitude for the UCHs. The broad range of mechanostability within the same protein family may be associated with the functional requirements for their differential malleabilities.

Identification ofEscherichia coliClpAP in regulating susceptibility to type VI secretion system-mediated attack byAgrobacterium tumefaciens

“…We took advantage of the widely adopted ClpP protease degradation assay using GFP-ssrA as the model substrate. Loss of GFP fluorescence is used as a reporter to monitor substrate degradation by ClpAP as a function of time (26,27). The results showed that over time, wild-type ClpP effectively degraded GFP-ssrA with a half-life of about 30 min (Fig.…”

“…GFP fluorescence based-degradation assays were carried out in PD buffer (25 mM HEPES, pH 7.5, 100 mM KCl, 25 mM MgCl2, 1 mM DTT, 10% glycerol) containing 3 µM GFP-ssrA as substrate and ATP regeneration system (16 mM creatine phosphatase, 0.32 mg/mL creatine kinase) as described previously (27). In brief, 0.1 μM ClpX6 and 0.3 μM ClpP14 or its variants were mixed at 30 °C and let stand for 2 min.…”

Knotted proteins: Tie etiquette in structural biology

“…There is a plethora of studies in the literature, both experimental and computational, which analyzed specific model systems and proposed several biological functions. It has been proposed that knotted proteins have enhanced thermal (20,26), mechanical (26,27,128) and structural (127) stabilities. Another possibility is that knots help shape and stabilize the active site of enzyme (129)(130)(131), and in some cases it has even been suggested that knots can control enzymatic (130) and signaling activity (132).…”

“…The realization that knotted proteins exist motivated the search for the functional advantages that knots may convey to their carriers. The analysis of specific model systems has put forward the idea that knots could enhance thermal (20,26) or mechanical stability (27), just to mention a few examples. However, a universal role for knots in proteins has not yet been identified.…”

Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

“…Interestingly, our substrate was able to be degraded even in the absence of the Rpn10 UIM domain, while in previous work from the Martin lab, degradation of an Rsp5-ubiquitinated GFP-containing substrate was completely dependent on Rpn10 37 . Possibly this substrate cannot bind to Rpn13 in a productively positioned manner or has different unfolding requirements than our substrate, as the topology and stability of a substrate may affect the force needed to unfold it 16,61,62 . Alternatively, reconstituted proteasome could be missing physiological modifications or proteasome-associated proteins (such as shuttle factors) that allow Rpn13 to be used productively or increase the proteasome's unfolding ability.…”