Designed Semisynthetic Protein Inhibitors of Ub/Ubl E1 Activating Enzymes

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 non-hydrolyzable 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 crosslink to SUMO E1 and Ub E1, respectively, in a cysteine nucleophile-dependent manner. These inhibitors limac@mskcc.org or tand@mskcc.org. The Ub/Ubl modifier is coupled by its C-terminal carboxylate to specific lysine sidechains on target proteins via an isopeptide bond. Initial steps in this process are catalyzed by a Ub/ Ubl activating enzyme (E1), which first adenylates the Ub/Ubl C-terminus to form a Ub/Ubl-AMP intermediate, then transfers the Ub/Ubl to a conserved cysteine on the E1 (Figure 1). The Ub/Ubl is then transthioesterified onto a cysteine side chain of a conjugating enzyme (E2), and finally transferred to a lysine side chain of the target protein, often mediated by a ligase (E3). Although structures of several E1s have been reported, 3,4 outstanding questions remain about the mechanisms of these reactions. First, E1s surprisingly crystallize with substrates bound in the active site, rather than the in situ-formed, presumably tighter binding acyl-AMP intermediate, 5 in contrast to other enyzmes that catalyze adenylation reactions. 6 Second, the conserved E1 cysteine that serves as the nucleophile in the thioesterification half-reaction is remote, >30 Å away from the adenylation active site. These observations suggest that additional conformational changes are required in both half-reactions. 3 To investigate these questions, we sought to develop mechanism-based inhibitors of E1s that could then be used in pivotal structural and biochemical studies. 7 Selective inhibitors would also be useful probes for dissecting E1 functions. We and others have used 5′-sulfonyladenosine-based small molecules to inhibit various mechanistically (but not structurally) related enzymes that catalyze adenylation reactions. 8 We envisioned that such inhibitor design strategies might also be effective for E1s and report herein the development of semisynthetic, C-terminally modified Ub/Ubl proteins as novel, ...

Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

“…To date inhibitors against two other E1 enzymes, the SUMO-1 activating enzyme (SAE) [125-127] and NEDD8 activating enzyme (NAE) have been developed [128]. NAE inhibition indirectly affects the UPS as NEDDylation is essential for the activation of the SCF (Skp, Cullin, F-box containing complex) Cullin-containing E3 complexes and thus represents a more specific approach than blocking UBE1.…”

Structural and Functional Insights to Ubiquitin-Like Protein Conjugation

“…This issue was resolved in part by utilizing synthetic adenylate analogs linked to SUMO and Ub to create structural analogs of the Ub/Ubl-adenylate. These adducts inhibit corresponding E1s, and the installation of an electrophilic trap at a position analogous to the carbonyl carbon of the C-terminal glycine successfully captured a cross-linked thioether E1 adduct that mimicks the tetrahedral intermediate during thioester bond formation (62). Subsequent structural studies using SUMO E1 with SUMO adenylate analogs revealed a new closed conformation wherein the E1 CYS domain rotates 130°, transiting the catalytic cysteine to a position proximal to the SUMO C-terminal adenylate analog that remains coordinated within a remodeled adenylate active site (Figure 1 c ) (70).…”

Biological Applications of Protein Splicing

“…A related concept has recently been applied to the study of E1 ubiquitin ligases (Lu et al, 2010; Olsen et al, 2010). These enzymes activate Ub and Ubls through adenylation (AMP) of their C-terminus followed by thioesterification of a conserved Cys residue in the enzyme.…”

“…These enzymes activate Ub and Ubls through adenylation (AMP) of their C-terminus followed by thioesterification of a conserved Cys residue in the enzyme. To probe the first half-reaction, EPL was used to generate a reversible inhibitor by incorporating a non-hydrolyzable analog of AMP, 5‘- O -sulfamoyladenosine (AMSN), at the C-terminus of Ub and the Ubl, SUMO (Lu et al, 2010). A similar EPL approach was used to obtain a covalent inhibitor of the second half-reaction, in this case by incorporating a vinyl-sulfonamide electrophilic trap into the moiety (AVSN).…”