Designed Semisynthetic Protein Inhibitors of Ub/Ubl E1 Activating Enzymes

摘要

Ubiquitin (Ub) and related ubiquitin-like (Ubl) proteins such asnSUMO serve as reversible, post-translational modifications ofnprotein substrates, impacting diverse cellular processes.n1,2nThe UbUbl modifier is coupled by its C-terminal carboxylate to specificnlysine side chains on target proteins via an isopeptide bond. Initialnsteps in this process are catalyzed by a Ub/Ubl activating enzymen(E1), which first adenylates the Ub/Ubl C-terminus to form a UbUbl-AMP intermediate and then transfers the Ub/Ubl to a conservedncysteine on the E1 (Figure 1). The Ub/Ubl is then transthioesterifiednonto a cysteine side chain of a conjugating enzyme (E2) and finallyntransferred to a lysine side chain of the target protein, often mediatednby a ligase (E3). Although structures of several E1s have beennreported,n3,4noutstanding questions remain about the mechanisms ofnthese reactions. First, E1s surprisingly crystallize with substratesnbound in the active site rather than the in situ-formed, presumablynmore tightly binding acyl-AMP intermediate,n5nin contrast to othernenyzmes that catalyze adenylation reactions.n6nSecond, the conservednE1 cysteine that serves as the nucleophile in the thioesterificationnhalf-reaction is remote, >30 Å away from the adenylation activensite. These observations suggest that additional conformationalnchanges are required in both half-reactions.n3nTo investigate thesenquestions, we sought to develop mechanism-based inhibitors of E1snthat could then be used in pivotal structural and biochemicalnstudies.n7nSelective inhibitors would also be useful probes forndissecting E1 functions. We and others have used 5′-sulfonylad-nenosine-based small molecules to inhibit various mechanisticallyn(but not structurally) related enzymes that catalyze adenylationnreactions.n8nWe envisioned that such inhibitor design strategies mightnalso be effective for E1s and report herein the development ofnsemisynthetic, C-terminally modified Ub/Ubl proteins as novel,nselective E1 inhibitors.