UFC1 reveals the multifactorial and plastic nature of oxyanion holes in E2 conjugating enzymes

Manoj Kumar, Sayanika Banerjee, Einav Cohen-Kfir, Marissa Basia Mitelberg, Suryakant Tiwari, Michail N. Isupov, Moshe Dessau and Reuven Wiener ()
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Manoj Kumar: Hebrew University-Hadassah Medical School
Sayanika Banerjee: Hebrew University-Hadassah Medical School
Einav Cohen-Kfir: Hebrew University-Hadassah Medical School
Marissa Basia Mitelberg: Hebrew University-Hadassah Medical School
Suryakant Tiwari: Bar-Ilan University
Michail N. Isupov: University of Exeter
Moshe Dessau: Bar-Ilan University
Reuven Wiener: Hebrew University-Hadassah Medical School

Nature Communications, 2025, vol. 16, issue 1, 1-17

Abstract: Abstract The conjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBL) to target proteins is a crucial post-translational modification that typically involves nucleophilic attack by a lysine on a charged E2 enzyme (E2~Ub/UBL), forming an oxyanion intermediate. Stabilizing this intermediate through an oxyanion hole is vital for progression of the reaction. Still, the mechanism of oxyanion stabilization in E2 enzymes remains unclear, although an asparagine residue in the conserved HPN motif of E2 enzymes was suggested to stabilize the oxyanion intermediate. Here, we study the E2 enzyme UFC1, which presents a TAK rather than an HPN motif. Crystal structures of UFC1 mutants, including one that mimics the oxyanion intermediate, combined with in vitro activity assays, suggest that UFC1 utilizes two distinct types of oxyanion holes, one that stabilizes the oxyanion intermediate during trans-ufmylation mediated by the E3 ligase, and another that stabilizes cis-driven auto-ufmylation. Our findings indicate that oxyanion stabilization is influenced by multiple factors, including C-alpha hydrogen bonding, and is adaptable, enabling different modes of action.

Date: 2025
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DOI: 10.1038/s41467-025-58826-y

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