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. 2010 Feb 17;132(6):1748-9.
doi: 10.1021/ja9088549.

Designed semisynthetic protein inhibitors of Ub/Ubl E1 activating enzymes

Xuequan Lu  1 , Shaun K Olsen, Allan D Capili, Justin S Cisar, Christopher D Lima, Derek S Tan
Affiliations

Designed semisynthetic protein inhibitors of Ub/Ubl E1 activating enzymes

Xuequan Lu et al. J Am Chem Soc. .

Abstract

Semisynthetic, mechanism-based protein inhibitors of ubiquitin (Ub) and ubiquitin-like modifier (Ubl) activating enzymes (E1s) have been developed to target E1-catalyzed adenylation and thioesterification of the Ub/Ubl C-terminus during the processes of protein SUMOylation and ubiquitination. The inhibitors were generated by intein-mediated expressed protein ligation using a truncated Ub/Ubl protein (SUMO residues 1-94; Ub residues 1-71) with a C-terminal thioester and synthetic tripeptides having a C-terminal adenosine analogue and an N-terminal cysteine residue. SUMO-AMSN (4a) and Ub-AMSN (4b) contain a sulfamide group as a nonhydrolyzable mimic of the phosphate group in the cognate Ub/Ubl-AMP adenylate intermediate in the first half-reaction, and these constructs selectively inhibit SUMO E1 and Ub E1, respectively, in a dose-dependent manner. SUMO-AVSN (5a) and Ub-AVSN (5b) contain an electrophilic vinyl sulfonamide designed to trap the incoming E1 cysteine nucleophile (Uba2 Cys173 in SUMO E1; Uba1 Cys593 in Ub E1) in the second half-reaction, and these constructs selectively, covalently, and stably cross-link to SUMO E1 and Ub E1, respectively, in a cysteine nucleophile-dependent manner. These inhibitors are powerful tools to probe outstanding mechanistic questions in E1 function and can also be used to study the biological functions of E1 enzymes.

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Figures

Figure 1
Figure 1
Ub/Ubl activating enzymes (E1) catalyze adenylation of a Ub/Ubl at its C-terminus followed by thioesterification at a conserved cysteine of the E1 (Uba2 Cys173 in SUMO E1; Uba1 Cys593 in Ub E1).
Figure 2
Figure 2
Semisynthesis of mechanism-based SUMO E1 and Ub E1 inhibitors (4, 5). (Ub/UblΔ = SUMO1–94; Ub1–71).
Figure 3
Figure 3
Inhibition of SUMO E1 (Sae1·Uba2) and Ub E1 (Uba1) by semisynthetic, C-terminally modified SUMO and Ub constructs (SDS-PAGE data). (a,b) SUMO-AMSN (4a) inhibits SUMO E1-S-SUMO thioester formation and Ub-AMSN (4b) inhibits Ub E1-S-Ub formation in a dose-dependent manner. (C) The constructs do not inhibit the non-cognate E1s. (d) SUMO-AVSN (5a) covalently crosslinks to SUMO E1 (Uba2 subunit) but not to Ub E1 (Uba1) and Ub-AVSN (5b) covalent crosslinks to Ub E1 (Uba1) but not to SUMO E1 (Uba2 subunit).
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References

    1. Johnson ES. Annu. Rev. Biochem. 2004;73:355–382. - PubMed
    2. Hershko A, Ciechanover A. Annu. Rev. Biochem. 1998;67:425–479. - PubMed

    1. The human SUMO-1 isoform, human Sae1·Uba2, S. pombe ubiquitin, and S. pombe Uba1 were used for all experiments herein, and are referred to as SUMO, SUMO E1, Ub, and Ub E1, respectively, for simplicity.

    1. (a) SUMO E1:Lois LM, Lima CD. EMBO J. 2005;24:439–451.(b) Nedd8 E1:Walden H, Podgorski MS, Huang DT, Miller DW, Howard RJ, Minor DL, Jr., Holton JM, Schulman BA. Mol. Cell. 2003;12:1427–1437.(c) Ubiquitin E1:Lee I, Schindelin H. Cell. 2008;134:268–278.

    1. Reviewed in:Capili AD, Lima CD. Curr. Opin. Struct. Biol. 2007;17:726–735.Dye BT, Schulman BA. Annu. Rev. Biophys. Biomol. Struct. 2007;36:131–150.

    1. The sole exception in the E1 family is MoeB, a bacterial biosynthetic enzyme consisting of only the adenylation domain of the corresponding eukaryotic enzymes, which has its cognate MoaD-AMP intermediate bound in its active site:Lake MW, Weubbens MM, Rajagopalan KV, Schindelin H. Nature. 2001;414:325–329.

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