Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome.
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 10-8s-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.
Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.|Institute of Biochemical Sciences, National Taiwan University, 10617, Taiwan.
Yun-Tzai Cloud
YC
Lee
Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.|Institute of Biochemical Sciences, National Taiwan University, 10617, Taiwan.
Chia-Yun
CY
Chang
Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
Szu-Yu
SY
Chen
Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
Yun-Ru
YR
Pan
Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.
Meng-Ru
MR
Ho
Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan.|Institute of Biochemical Sciences, National Taiwan University, 10617, Taiwan.
Shang-Te Danny
SD
Hsu
D019277:Entropy; D006801:Humans; D056004:Molecular Dynamics Simulation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D000072417:Protein Domains; D058767:Protein Unfolding; D043222:Ubiquitin Thiolesterase / Q000737:chemistry* / Q000378:metabolism
C421245:UCHL5 protein, human; D043222:Ubiquitin Thiolesterase; D046988:Proteasome Endopeptidase Complex