Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome

Abstract

Human ubiquitin C-terminal hydrolyase UCH-L5 is a topologically knotted deubiquitinase that is activated upon binding to the proteasome subunit Rpn13. The length of its intrinsically disordered cross-over loop is essential for substrate recognition. Here, we showed that the catalytic domain of UCH-L5 exhibits higher equilibrium folding stability with an unfolding rate on the scale of 10−8 s−1, over four orders of magnitudes slower than its paralogs, namely UCH-L1 and -L3, which have shorter cross-over loops. NMR relaxation dynamics analysis confirmed the intrinsic disorder of the cross-over loop. Hydrogen deuterium exchange analysis further revealed a positive correlation between the length of the cross-over loop and the degree of local fluctuations, despite UCH-L5 being thermodynamically and kinetically more stable than the shorter UCHs. Considering the role of UCH-L5 in removing K48-linked ubiquitin to prevent proteasomal degradation of ubiquitinated substrates, our findings offered mechanistic insights into the evolution of UCH-L5. Compared to its paralogs, it is entropically stabilized to withstand mechanical unfolding by the proteasome while maintaining structural plasticity. It can therefore accommodate a broad range of substrate geometries at the cost of unfavourable entropic loss.

The exclusive effects of chaperonin on the behavior of proteins with 52 knot

“…The landscape of successful folding pathways that leads to the correctly knotted native conformation is observed for each of the proteins, in both conditions through our simulations. As the folding of all UCHs is similar [ 31 ], we concentrated our study on the most commonly analyzed protein—UCH-L1 (PDB code 3IRT), comparing results with other UCHs when needed. Our results naturally split into three parts: description of folding/unfolding landscape, kinetics of the process and an analysis of the random and short-lived knots.…”

“…Folding of UCHs is known to follow two parallel pathways [ 30 , 31 ]. In our simulations, data analysis of time evolution of the topology revealed also two, topologically distinct pathways, shown schematically in Fig 2 .…”

“…The UCHs are known to fold and refold along two parallel pathways, each featuring one slow and one fast phase [ 30 , 31 , 52 ]. Therefore, to correlate our model with experimental results we recreated the chevron plot for UCH-L1, with the temperature as a denaturant.…”

“…However, any theoretical results have to be confronted with the experimental data concerning UCHs folding. It has been shown already that UCHs can fold and refold reversibly in two parallel pathways, each consisting of one slow and one fast phase, as determined from chevron plot [ 30 , 31 ]. Despite a common mechanism, folding processes of different UCHs is characterized by different kinetic parameters.…”

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

“…While several theoretical models have been proposed to explain how protein knotting may be achieved 13 – 20 , it remains very challenging to experimentally visualize the knotting events along the folding pathway(s) of a given protein 2 , 21 . Over the years, a series of systematic experimental analyses on the folding dynamics and kinetics of a broad range of knotted proteins have been reported 22 – 29 , and a recurrent feature is the presence of highly populated folding intermediates, indicating rugged free energy landscapes associated with the folding knotted proteins. The folding stabilities of knotted proteins have so far been assessed exclusively by chemical denaturation (using urea or guanidium hydrochloride (GdnHCl) as denaturants) and to a lesser extent by thermal denaturation.…”