Intrinsic disorder drives N-terminal ubiquitination by Ube2w

Abstract

Ubiquitination of the αN-terminus of protein substrates has been reported sporadically over the past twenty years. However the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the ubiquitin-conjugating enzyme (E2) Ube2w employs a novel mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N-termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike any other E2, in which its C-terminus is partly disordered and flexible to accommodate variable substrate N-termini. Flexibility of the substrate is critical for recognition by Ube2w and point mutations in, or removal of, the flexible C-terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal-Ubiquitome in cells.

Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21

“…These results are in good agreement with previous reports of cooperation between Ube2W and Ube2N/Ube2V2 (12,22) and the ability of Ube2W to mediate specific N-terminal ubiquitination of substrates (22)(23)(24). In the absence of Ube2W, Ube2N/Ube2V2 and TRIM21 synthesize unanchored Ub-63 Ub in vitro.…”

“…2E, lane 3). (22)(23)(24). To assess whether TRIM21 is also ubiquitinated by Ube2W independent of lysine residues in vitro, we purified TRIM21 RING-Box proteins bearing lysine-to-arginine substitutions at all six lysine residues in TRIM21 RING-Box : three in the RING domain (Lys45, Lys61, Lys77) and three in the B-Box2 domain (Lys105, Lys108, Lys119).…”

Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems

“…7B) (27), further suggesting an accumulation of relatively disordered proteins in Ube2w KO testis. Although direct UBE2W substrates were not identified in our study, these results are consistent with the in vitro evidence that UBE2W preferentially ubiquitinates disordered substrates (21). This preference of UBE2W, however, is believed to be most critical for disorder at the N terminus.…”

“…UBE2W may preferentially target substrates with intrinsically unfolded N termini (21), but its expression and function in vivo are poorly understood. Our data reveal widespread UBE2W expression in the mouse, particularly of UBE2W isoform 1, with protein translation beginning at the second predicted Kozak sequence.…”

“…It can function with various ubiquitin ligases, including the C terminus of Hsc-70-interacting protein and the BRCA1-BARD1 complex (18 -20), to monoubiquitinate select substrates at their N termini with an apparent preference for substrates having intrinsically disordered N termini (21). In addition, monoubiquitination of FANCD2, which can be mediated by UBE2W (22), is critical for activation of the Fanconi anemia tumor suppressor pathway in the DNA damage response (11,23), but the role of UBE2W in DNA damage pathways is not well studied.…”