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. 2013 Feb 27;135(8):2867-70.
doi: 10.1021/ja309802n. Epub 2013 Feb 15.

On terminal alkynes that can react with active-site cysteine nucleophiles in proteases

Reggy Ekkebus  1 , Sander I van Kasteren, Yogesh Kulathu, Arjen Scholten, Ilana Berlin, Paul P Geurink, Annemieke de Jong, Soenita Goerdayal, Jacques Neefjes, Albert J R Heck, David Komander, Huib Ovaa
Affiliations
Free PMC article

On terminal alkynes that can react with active-site cysteine nucleophiles in proteases

Reggy Ekkebus et al. J Am Chem Soc. .
Free PMC article

Abstract

Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

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Figures

Figure 1
Figure 1
(A) Ubiquitin functionalized with propargylamine replacing Gly76. (B) Fluorescence polarization-based substrate turnover assay measuring UCHL3 activity, showing Ub-Prg as 105 times more powerful an inhibitor than Ub. Dotted line represents the concentration of UCHL3 (60 pM) used. (C) Deconvoluted mass of UCHL3 (calcd monoisotopic mass, 26 166 Da) before/after reaction with Ub-Prg. A mass increase of 8544 Da is observed, corresponding to one Ub-Prg molecule.
Figure 2
Figure 2
Structure of vOTU (blue) in complex with Ub-Prg (yellow). (A) Electron density maps (blue represents 2|Fo| – |Fc| contoured at 1σ; green represents |Fo| – |Fc at 3σ) covering the catalytic Cys of vOTU and the C-terminus of Ub-Prg, before (top) and after refinement (bottom) with the vinyl thioether linker. (B) Reaction between vOTU and Ub-Prg (top) and representation of the reaction product as observed by X-ray crystallography (bottom), showing the vinyl thioether linkage in the Ub-Prg complexed structure.
Figure 3
Figure 3
SDS-PAGE gel showing reaction of Ub-Prg derivatives. R1 = N-terminally TMR-labeled Ub1–75, R2 = unlabeled Ub1–75, and R3 = Ub1–74. Only probes 6, 7, and Ub-Prg show significant reactivity toward UCHL3.
Figure 4
Figure 4
(A) SDS-PAGE analysis of in vitro reaction of three different classes of DUBs with Ub-Prg. (B) GFP fusions of DUBs from the USP and OTU-clades were transfected in MelJuSo cells, and their reaction with Ub-Prg was visualized using anti-GFP Western blot. DUBs annotated with -CS are catalytic cysteine-to-serine mutants. For images of direct fluorescence scans see Figure S8. (C) Comparison of DUB labeling between TMR-Ub-VME and TMR-Ub-Prg. Third and fifth lanes are pretreated with unlabeled Ub-Prg and Ub-VME, respectively. Labeled lysates were analyzed using SDS-PAGE, and tagged DUBs were visualized by fluorescence scanning. An overexposed fluorescent image showing DUBs 3–5 is available as Figure S10.
Figure 5
Figure 5
Quantification of DUBs precipitated from EL-4 lysate after on-bead reaction with Ub-Prg. Ratios of ion intensities of proteins retrieved from Ub-Prg-resin pulldown vs pulldown with a control resin are shown.
Figure 6
Figure 6
Labeling of recombinant caspase-1 with alkyne-based caspase-1 probe analyzed by fluorescent scanning of SDS-PAGE gel. Recombinant caspase-1 was labeled with two different lengths of caspase probe (left) and doped in U937 lysate (right), showing selective labeling of caspase-1 in lysate.
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