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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.

SUBMITTER: Lee YC

PROVIDER: S-EPMC5364529 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Lee Yun-Tzai Cloud YC Chang Chia-Yun CY Chen Szu-Yu SY Pan Yun-Ru YR Ho Meng-Ru MR Hsu Shang-Te Danny SD

Scientific reports 20170324

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<sup>-8</sup> s<sup>-1</sup>, over four orders of magnitudes slower than its paralogs, namely UCH-L1 and -L3, w ...[more]

PMID: 28338014

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Project description:The osmolyte trimethylamine N-oxide (TMAO) accumulates in the cell in response to osmotic stress and increases the thermodynamic stability of folded proteins. To understand the mechanism of TMAO induced stabilization of folded protein states, we systematically investigated the action of TMAO on several model dipeptides (leucine, L(2), serine, S(2), glutamine, Q(2), lysine, K(2), and glycine, G(2)) in order to elucidate the effect of residue-specific TMAO interactions on small fragments of solvent-exposed conformations of the denatured states of proteins. We find that TMAO preferentially hydrogen bonds with the exposed dipeptide backbone but generally not with nonpolar or polar side chains. However, interactions with the positively charged Lys are substantially greater than with the backbone. The dipeptide G(2) is a useful model of the pure amide backbone; interacts with TMAO by forming a hydrogen bond between the amide nitrogen and the oxygen in TMAO. In contrast, TMAO is depleted from the protein backbone in the hexapeptide G(6), which shows that the length of the polypeptide chain is relevant in aqueous TMAO solutions. These simulations lead to the hypothesis that TMAO-induced stabilization of proteins and peptides is a consequence of depletion of the solute from the protein surface provided intramolecular interactions are more favorable than those between TMAO and the backbone. To test our hypothesis, we performed additional simulations of the action of TMAO on an intrinsically disordered Aβ(16-22) (KLVFFAE) monomer. In the absence of TMAO, Aβ(16-22) is a disordered random coil. However, in aqueous TMAO solution, Aβ(16-22) monomer samples compact conformations. A transition from random coil to α-helical secondary structure is observed at high TMAO concentrations. The coil to α-helix transition is highly cooperative especially considering the small number of residues in Aβ(16-22). Our work highlights the potential similarities between the action of TMAO on long polypeptide chains and entropic stabilization of proteins in a crowded environment due to excluded volume interactions. In this sense, the chemical chaperone TMAO is a nanocrowding particle.

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Dataset Information

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

Publications

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

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