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

Abstract

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 β-sheet is highly structured and α-helix 3, which is partially formed, packs against it. In contrast to the stable β-sheet core, the peripheral α-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 α-helices 3 and 7 and the degree of structure in β-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 β-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 52-knot occurs late on the folding pathway.

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

“…2). For WT UCH-L1, these phases have been shown to correspond to transitions between the native state and the two intermediate states (29). We assume that this is also the case for all of the variants, and the data are consistent with this assumption.…”

“…The data were fit to either a two-or three-state unfolding model depending on the probe and variant used (see Supporting Materials and Methods) for further details. The refolding/unfolding kinetic measurements were carried out as previously described (29). Details regarding the methods used can be found in Supporting Materials and Methods.…”

“…The intrinsic Trp fluorescence was used to probe changes in tertiary structure. Both single-[urea]-jump unfolding/refolding kinetics and interrupted-refolding kinetics were recently performed in experiments with WT UCH-L1 (29). The results from those experiments were used to propose a kinetic scheme for folding ( Fig.…”

“…a WT data were taken from an earlier investigation (29) and analyzed using a simple two-state model (Eq. S8).…”

“…Researchers have investigated wildtype (WT) UCH-L1 using a range of different techniques. For example, the fluorescence of the single tryptophan, Trp26, was used to probe changes in tertiary structure in both thermodynamic and kinetic studies (28,29). These studies showed that UCH-L1 unfolds reversibly under equilibrium conditions using urea as a denaturant, and that at least one stable intermediate state (I) is populated between the native (N) and denatured (D) states (28).…”