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

Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D, Ovaa H - J. Am. Chem. Soc. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

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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Structure of vOTU (blue) in complex withUb-Prg (yellow). (A) Electrondensity maps (blue represents 2/Fo/ – /Fc/ contoured at 1σ; green represents /Fo/ – /Fc at 3σ) covering the catalytic Cys of vOTU andthe C-terminus of Ub-Prg, before (top) and after refinement (bottom)with the vinyl thioether linker. (B) Reaction between vOTU andUb-Prg (top) and representation of the reaction product as observedby X-ray crystallography (bottom), showing the vinyl thioetherlinkage in the Ub-Prg complexed structure.
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fig2: Structure of vOTU (blue) in complex withUb-Prg (yellow). (A) Electrondensity maps (blue represents 2/Fo/ – /Fc/ contoured at 1σ; green represents /Fo/ – /Fc at 3σ) covering the catalytic Cys of vOTU andthe C-terminus of Ub-Prg, before (top) and after refinement (bottom)with the vinyl thioether linker. (B) Reaction between vOTU andUb-Prg (top) and representation of the reaction product as observedby X-ray crystallography (bottom), showing the vinyl thioetherlinkage in the Ub-Prg complexed structure.

Mentions: To determine the reaction rate, we performed an in vitro time course experiment (FigureS3). UCHL3showed quantitative reaction with Ub-Prg within 1 min, similar tothe rate previously reported for the Ub-based DUB-probe, Ub vinylmethyl ester (Ub-VME).11 Reaction betweenUCHL3 and Ub-Prg yielded a product equal in mass to the sum of bothreactants. The acid lability of the purified UCHL3·Ub-Prg complex(Figure S4) suggested the formation ofa vinyl thioether linkage. The nature ofthe linkage formed was confirmed by solving the crystal structureof a DUB in complex with Ub-Prg. The viral ovarian tumor DUB (vOTU)encoded by Crimean Congo hemorrhagic fever virus (CCHFV)12 was reacted with Ub-Prg. The resulting complexwas crystallized and its structure determined at 2.3 Å resolution(Figure 2A, Table S1). The refined structure closely resembles previously determinedvOTU·Ub complexes13 (rmsd = 0.4–0.6Å2). Refinement of the complex structure excludingthe propargyl group at the C-terminus of Ub yielded positive differenceelectron density (/Fo/ – /Fc) connecting Gly75 to the catalytic Cys40 residueof vOTU (Figure 2A). The electron density obtainedunambiguously revealed the attachment of the Cys thiol atom to thequaternary carbon in Ub-Prg (Figure 2B), confirminga vinyl thioether linkage.


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

Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D, Ovaa H - J. Am. Chem. Soc. (2013)

Structure of vOTU (blue) in complex withUb-Prg (yellow). (A) Electrondensity maps (blue represents 2/Fo/ – /Fc/ contoured at 1σ; green represents /Fo/ – /Fc at 3σ) covering the catalytic Cys of vOTU andthe C-terminus of Ub-Prg, before (top) and after refinement (bottom)with the vinyl thioether linker. (B) Reaction between vOTU andUb-Prg (top) and representation of the reaction product as observedby X-ray crystallography (bottom), showing the vinyl thioetherlinkage in the Ub-Prg complexed structure.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585465&req=5

fig2: Structure of vOTU (blue) in complex withUb-Prg (yellow). (A) Electrondensity maps (blue represents 2/Fo/ – /Fc/ contoured at 1σ; green represents /Fo/ – /Fc at 3σ) covering the catalytic Cys of vOTU andthe C-terminus of Ub-Prg, before (top) and after refinement (bottom)with the vinyl thioether linker. (B) Reaction between vOTU andUb-Prg (top) and representation of the reaction product as observedby X-ray crystallography (bottom), showing the vinyl thioetherlinkage in the Ub-Prg complexed structure.
Mentions: To determine the reaction rate, we performed an in vitro time course experiment (FigureS3). UCHL3showed quantitative reaction with Ub-Prg within 1 min, similar tothe rate previously reported for the Ub-based DUB-probe, Ub vinylmethyl ester (Ub-VME).11 Reaction betweenUCHL3 and Ub-Prg yielded a product equal in mass to the sum of bothreactants. The acid lability of the purified UCHL3·Ub-Prg complex(Figure S4) suggested the formation ofa vinyl thioether linkage. The nature ofthe linkage formed was confirmed by solving the crystal structureof a DUB in complex with Ub-Prg. The viral ovarian tumor DUB (vOTU)encoded by Crimean Congo hemorrhagic fever virus (CCHFV)12 was reacted with Ub-Prg. The resulting complexwas crystallized and its structure determined at 2.3 Å resolution(Figure 2A, Table S1). The refined structure closely resembles previously determinedvOTU·Ub complexes13 (rmsd = 0.4–0.6Å2). Refinement of the complex structure excludingthe propargyl group at the C-terminus of Ub yielded positive differenceelectron density (/Fo/ – /Fc) connecting Gly75 to the catalytic Cys40 residueof vOTU (Figure 2A). The electron density obtainedunambiguously revealed the attachment of the Cys thiol atom to thequaternary carbon in Ub-Prg (Figure 2B), confirminga vinyl thioether linkage.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus