Design and Semisynthesis of Biselectrophile-Functionalized Ubiquitin Probes To Investigate Transthioesterification Reactions

Abstract

Ubiquitin (Ub) regulates a wide array of cellular processes through post-translational modification of protein substrates. Ub is conjugated at its C-terminus to target proteins via an enzymatic cascade in which covalently bound Ub thioesters are transferred from E1 activating enzymes to E2 conjugating enzymes, and then to certain E3 protein ligases. These transthioesterification reactions proceed via transient tetrahedral intermediates. A variety of chemical strategies have been used to capture E1-Ub-E2 and E2-Ub-E3 mimics, but these introduce modifications that disrupt atomic spacing at the linkage point relative to the native tetrahedral intermediate. We have developed a biselectrophilic PSAN warhead that can be installed in place of the conserved C-terminal glycine in Ub and used to form ternary protein complexes linked via cyanomethyldithioacetals that closely mimic the native tetrahedral intermediates. Investigation of the reactivity of the warhead and substituted analogues led to an effective semisynthetic route to Ub^-1-PSAN, which was used to form a ternary E1-Ub*-E2 complex as a mimic of the transthioesterification intermediate.

Figure 1

Ubiquitin conjugation cascade and probes. (a) Ub is transthioesterified from E1 activating enzymes to E2 conjugating enzymes and from E2s to thioester-forming E3 protein ligases via tetrahedral intermediates 3 and 5 (^†RING-type E3s noncovalently catalyze Ub transfer from E2∼Ub thioesters directly to targets (4 → 7)). (b) Biselectrophilic Ub^–1-PSAN probe used herein to form an E1–Ub*–E2 conjugate as mimic of tetrahedral intermediate 3 (Ub* = cyanomethyl-modified Ub). (c) Structures of related small-molecule biselectrophiles.

Figure 2

Synthesis and reactivity of XSAN warheads. (a) Synthesis of warheads 14 and model reactions with a cysteine nucleophile (blue). (b) Formation of cross-linked E1–X–E2 complexes with biselectrophiles (X). E1 = Schizosaccharomyces pombe Uba1 (50 nM); E2 = S. pombe Ubc13; X = biselectrophile; calculated MW: E1–X–E2 = 129 kDa, E1 = 112 kDa; SDS-PAGE, Sypro Ruby stain. (c) Attempted synthesis of Ub^–1-PSAN (18) by aminolysis of Ub^–1 MESNa thioester 17 (S. pombe Ub[1–75]; MESNa = mercaptoethanesulfonate, sodium salt) or an in situ-generated Ub^–1 acyl azide 20.

Figure 3

Synthesis and reactivity of Gly-XSAN warheads. Second-order rate constant for Ac-Cys-OMe addition to E-23a and Z-23a obtained via pseudo-first-order kinetic analysis with k_obs plotted against Ac-Cys-OMe concentration.

Figure 4

Synthesis and reactivity of Ub^–1-XSAN probes. (a) Synthesis of Ub^–1-XSAN probes 18a–c¹² and reaction with an E2 to form E2–Ub* intermediate 28, then with an E1 to form E1–Ub*–E2 ternary complex 29 (Ub* = cyanomethylidiene- or cyanomethyl-modified Ub). (b) Ub^–1-PSAN 18a (0–500 μM) reacts with wild-type E2 (S. pombe Ubc13, 200 μΜ), but not an C86A mutant that lacks the catalytic cysteine, to form E2–Ub* intermediate 28. (c) Reaction of Ub^–1-PSAN 18a (400 μM) with other E2s (200 μM) to form E2–Ub* intermediates 28. (d) Reaction of the unpurified E2–Ub* intermediate 28 (S. pombe Ubc13, 60 μM total input to Ub^–1-PSAN conjugation) with an E1 (S. pombe Uba1, 10 μM) forms E1–Ub*–E2 complex 29. The tetrahedral intermediate mimic 29 (lanes 4) is not cleaved by β-mercaptoethanol (right), in contrast to E1–E2 disulfide and E1∼Ub thioester controls (lanes 2,3), and is not formed with an E1 C593A mutant lacking the catalytic cysteine. SDS-PAGE, Coomassie stain for all gels.