The Knotted Protein UCH-L1 Exhibits Partially Unfolded Forms under Native Conditions that Share Common Structural Features with Its Kinetic Folding Intermediates

JOURNAL OF MOLECULAR BIOLOGY

卷:428

Article

Lou, Shih-Chi^1,2  Wetzel, Svava¹  Zhang, Hongyu¹  Crone, Elizabeth W.¹  Lee, Yun-Tzai^2,3  Jackson, Sophie E.¹  Hsu, Shang-Te Danny^1,2,3 

[1] Univ Cambridge, Dept Chem, Lensfield Rd, Cambridge CB2 1EW, England

[2] Acad Sinica, Inst Biol Chem, 128,Sect 2,Acad Rd, Taipei 11529, Taiwan

[3] Natl Taiwan Univ, Inst Biochem Sci, 1,Sect 4,Roosevelt Rd, Taipei 106, Taiwan

关键词: knotted protein;    ubiquitin C-terminal hydrolase;    folding pathway;    partially unfolded form;    folding intermediate;   

DOI  :  10.1016/j.jmb.2016.04.002

来源: Elsevier

【 摘 要 】

The human ubiquitin C-terminal hydrolase, UCH-L1, is an abundant neuronal deubiquitinase that is associated with Parkinson's disease. It contains a complex Gordian knot topology formed by the polypeptide chain alone. Using a combination of fluorescence-based kinetic measurements, we show that UCH-L1 has two distinct kinetic folding intermediates that are transiently populated on parallel pathways between the denatured and native states. NMR hydrogen-deuterium exchange (HDX) experiments indicate the presence of partially unfolded forms (PUFs) of UCH-L1 under native conditions. HDX measurements as a function of urea concentration were used to establish the structure of the PUFs and pulse-labelled HDX NMR was used to show that the PUFs and the folding intermediates are likely the same species. In both cases, a similar stable core encompassing most of the central beta-sheet is highly structured and alpha-helix 3, which is partially formed, packs against it. In contrast to the stable beta-sheet core, the peripheral alpha-helices display significant local fluctuations leading to rapid exchange. The results also suggest that the main difference between the two kinetic intermediates is structure and packing of alpha-helices 3 and 7 and the degree of structure in beta-strand 5. Together, the fluorescence and NMR results establish that UCH-L1 neither folds through a continuum of pathways nor by a single discrete pathway. Its folding is complex, the beta-sheet core forms early and is present in both intermediate states, and the rate-limiting step which is likely to involve the threading of the chain to form the 5(2)-knot occurs late on the folding pathway. (C) 2016 Elsevier Ltd. All rights reserved.

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