Enzymatic production of mono-ubiquitinated proteins for structural studies: The example of the Josephin domain of ataxin-3

Abstract

Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration. However, despite the undoubted biological importance, understanding the molecular basis of how ubiquitination regulates different pathways has up to now been strongly limited by the difficulty of producing the amounts of highly homogeneous samples that are needed for a structural characterization by X-ray crystallography and/or NMR. Here, we report on the production of milligrams of highly pure Josephin mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. We demonstrate that the samples produced with the described method are correctly folded and suitable for structural studies. The protocol allows facile selective labelling of the components. Our results provide an important proof-of-concept that may pave the way to new approaches to the in vitro study of ubiquitinated proteins.

Keywords: ATP, adenosine triphosphate; DTT, dithiothreitol; DUB, deubiquitinating enzyme; Deubiquitinating enzyme; GST, glutathione-S-transferase; HSQC, heteronuclear single quantum coherence; IAA, iodoacetamide; Isopeptide bond; JosK117-only, Josephin mutant in which all lysines but K117 are mutated; Josephin; MS/MS tandem, mass spectrometry; Machado–Joseph disease; NMR, nuclear magnetic resonance; PDB, Protein Data Bank; Post-translational modification; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Spinocerebellar ataxia type 3; Tris–HCl, 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride; Ubiquitin.

Graphical abstract

Fig. 1

Scheme of the protocol set up for the production of mono-ubiquitinated JosK117-only.

Fig. 2

In vitro ubiquitination of JosK117-only. All reactions are monitored at t = 0, 3 and 20 h. Lanes 1–3: reaction using commercial enzymes (0.16 μM E1, 8 μM UbcH5a and 1 μM CHIP), 50 μM ubiquitin and 12 μM JosK117-only at room temperature [27]. Lanes 4–6: reaction using commercial enzymes (1 μM E1, 8 μM UbcH5a, 8 μM CHIP), 250 μM ubiquitin and 50 μM JosK117-only. Lanes 7–9: reaction using enzymes prepared in the lab at the final concentrations as in lanes 4–6. Lanes 10–12: reaction using Josephin pre-treated with IAA. Lane 13: purified mono-ubiquitinated JosK117-only. Lane 14: molecular weight marker. Di-ubiquitin and poly-ubiquitin chains are indicated with arrows. The bands of E1, E2 and E3 enzymes are indicated with annotation below each corresponding band. The Addgene clone of UbcH5a used for the production in the lab comprise an initial sequence deriving from cloning which explains the higher MW with respect to the commercial enzyme.

Fig. 3

Anion exchange purification of mono-ubiquitinated JosK117-only. Absorbance at 280 nm (blue), 250 nm (red) and percentage of buffer B (1 M NaCl) are reported. Mono-ubiquitinated JosK117-only elutes at ∼47 ml. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

MS analysis of JosK117-only. Top: MS spectrum of mono-ubiquitinated JosK117-only after trypsin digestion. The left arrow corresponds to Josephin GAMESIFHER N-terminal peptide, the right arrow indicates the mass of amino acids 111–124 including the ubiquitin C-terminal GG dipeptide covalently attached to lysine 117. Bottom: fragmentation with MS/MS of the peptide containing lysine 117.

Fig. 5

Comparison of NMR spectra of different Josephin samples. (A) ¹⁵N HSQC spectrum of labelled Josephin wild-type (in cyan) superimposed to the spectrum of labelled JosK117-only (in blue). (B) ¹⁵N HSQC spectrum of labelled JosK117-only covalently linked to unlabelled ubiquitin (in green) superimposed to the spectrum of labelled JosK117-only (in blue). (C) ¹⁵N HSQC spectrum of labelled ubiquitin covalently linked to unlabelled JosK117-only (in green) superimposed to the spectrum of labelled ubiquitin (in blue).The samples contain 300 μM proteins in 20 mM Na phosphate pH 6.5, 2 mM DTT. The spectra were recorded at 700 MHz and 25 °C. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)