A novel mechanism of ubiquitination suggests direct interaction between UBA1 and E3

Authors

  • VV Courouble
  • W Yan
  • F Shengyun
  • CA LeClair
  • D Tao

Abstract

Ubiquitination is a fundamental biological process that regulates numerous essential cellular functions via an enzymatic cascade involving E1 ubiquitin-activating enzymes, over two dozen E2 ubiquitin-conjugating enzymes, and ~600 E3 ubiquitin ligases. Impaired activity of UBA1, the primary E1 enzyme, leading to abnormal or decreased ubiquitination is responsible for driving many human diseases. We identified auranofin (AF), an approved drug used to treat rheumatoid arthritis, as the first UBA1 activity enhancer. AF binds to the UBA1 ubiquitin fusion domain to increase E2 interaction thus facilitating ubiquitin trans-thioesterification of E2 and increasing E3 activities. Interactome analysis found UBA1 interacts with twelve E2s and six E3s, which conflicts with the current dogma that ubiquitin-charged E2 dissociates from E1 before binding to spatially localized E3s. However, this model is bioenergetically unfavorable and lacks efficiency. As such, we propose UBA1 and E2 simultaneously interact with E3s to form a ternary complex to enable contemporaneous ubiquitin activation, ubiquitin charging to E2, and catalyzing substrate ubiquitination. Utilizing cross-linking mass spectrometry (XL-MS) and computational modeling, the first structural models of the UBA1-UBE2G2-Aup1 ternary complex were generated. The data revealed extensive intramolecular crosslinks in the cytosolic tail of Aup1, expected inter-protein crosslinks between Aup1 and UBE2G2, and a novel inter-protein crosslink between Aup1 and UBA1. Mapping the crosslinks onto the ternary models showed median distances at or below the crosslinker threshold of 31 Å, which supports that our models represent physiologically relevant conformations of the ternary complex. This was further validated by biochemical and cell biology experiments.

Scientific Focus Area: Structural Biology

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