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.

Recent studies on the mechanisms by which topologically knotted proteins attain their natively knotted structures have intrigued theoretical and experimental biophysicists. Of particular interest is the finding that YibK and YbeA, two small trefoil knotted proteins, remain topologically knotted in their chemically denatured states. Using small-angle X-ray scattering (SAXS), we examine whether these chemically denatured knotted proteins are different from typical random coils. By revisiting the scaling law of radius of gyration (Rg) as a function of polypeptide chain length for chemically denatured proteins and natively folded proteins, we find that the chemically denatured knotted proteins in fact follow the same random coil-like behavior, suggesting that the formation of topological protein knots do not necessarily require global compaction while the loosely knotted polypeptide chains are capable of maintaining the correct chirality without defined secondary or tertiary structures.

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.