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

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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.
Funding Information
  • Howard Hughes Medical Institute
  • National Science Foundation Graduate Research Fellowship Program (GFRP 2015190598)
  • National Cancer Institute (CCSG P30 CA008748)
  • Commonwealth Foundation
  • Division of Microbiology and Infectious Diseases (P30 CA008748, R01 AI118224)
  • National Institute of General Medical Sciences (R35 GM118080)
  • MSK Center for Experimental Therapeutics

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