Discovery and characterization of noncanonical E2-conjugating enzymes

Abstract

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.

Fig. 1.. The E2 MALDI-TOF discharge assay identifies E2 noncanonical activity.

Ubiquitin enzymes (E1 and E2) were incubated with adenosine triphosphate (ATP)/MgCl₂ solution and the indicated nucleophiles. Samples were analyzed by MALDI-TOF MS (A). Relative quantification of E2 discharge activity on ubiquitin is obtained by use of an internal standard (¹⁵N Ubiquitin) (B). Twenty-three E2s were tested for their ability to discharge ubiquitin on the indicated nucleophiles [(C) heatmap data are reported as the average of at least two technical replicates]. The ability of UBE2D3, UBE2Q1, UBE2Q2, and UBE2J2 to mediate ubiquitin discharge on lysine, serine, and threonine was further validated through a time course experiment [(D) N = 3, data are shown as means ± SD from three replicates].

Fig. 2.. UBE2Q1 ubiquitylates B4GALT1 CD in vitro.

UBE2Q1 autoubiquitylation species are sensitive to hydroxylamine (NH₂OH) and sodium hydroxide treatment (NaOH) (A). Schematic of B4GALT1 CD peptides synthesized in this study (B). Full-length UBE2Q1 directly ubiquitylates B4GALT1 on cysteine and serine residues (C). UBE2Q1 and UBE2Q2 schematic representation (D). UBE2Q1 UBC-domain is sufficient to directly ubiquitylate B4GALT1 CD (E). SDS–polyacrylamide gel electrophoresis gels are representative of three independent experiments.

Fig. 3.. Model of UBE2Q1~Ub in closed conformation and biochemical validation.

Side chains of residues at the interface between ubiquitin and UBE2Q1 highlighted in inset (A). UBE2Q1 residues interacting with ubiquitin were mutated and tested for discharge on B4GALT1 peptide1 (B). Residues of the ubiquitin Ile⁴⁴ patch were mutated as indicated and tested for UBE2Q1-mediated discharge on B4GALT1 peptide 1 (C). Data are representative of three independent experiments.

Fig. 4.. Identification of UBE2Q1 activity determinants.

Model of UBE2Q1 residues Tyr³⁴³ (Y343), Trp⁴¹⁴ (W414), and His⁴⁰⁹ (H409) side chains interacting with ubiquitin C terminus (A). Indicated UBE2Q1 mutants were tested for their ability to ubiquitylate B4GALT1 peptide 1 [(B) data are representative of three independent experiments] or to discharge on the indicated nucleophiles using the MALDI-TOF MS E2 discharge assay (C); bars represent the mean of four technical replicates.

Fig. 5.. B4GALT1 CD is highly conserved.

Sequence alignment of B4GALT1 CD domain in mammals (A). Serine residues were systematically mutated into alanine, lysine, or threonine and tested for UBE2Q1 mono-ubiquitylation in nonreducing (B) and reducing conditions (C). Data are representative of three independent experiments.

Fig. 6.. Relative expression of E2s in human and mouse tissues.

E2 in vivo expression profiling obtained by data mining of publicly available quantitative proteomic dataset of 32 human tissues (A). Relative expression of UBE2Q1 in indicated human tissues (B). Western blot validation of UBE2Q1 expression in the indicated mouse tissues [(C) N = 3 biological replicates].

Fig. 7.. UBE2J2 ubiquitylates serine and cysteine residues.

UBE2J2 facilitates the attachment of ubiquitin molecules to serine and cysteine amino acid residues within B4GALT1 peptide 1. UBE2J2-mediated ubiquitylation of B4GALT1 peptide 1 in nonreducing (A) and reducing (B) conditions. Specific mutation to WT B4GALT1 peptide 1 sequence highlighted in red [(A) and (B)]. Ile⁴⁴ patch ubiquitin mutations do not abolish UBE2J2 activity toward B4GALT1 peptide 1 (C). Data are representative of three independent experiments.

Fig. 8.. Identification of UBE2J2 activity determinants.

Predicted interaction between UBE2J2 and ubiquitin in an open conformation model. Inset, side chains of residues relevant for enzymatic activity (A). Indicated UBE2J2 mutants were tested using B4GALT1 peptide 1 ubiquitylation [(B) data are representative of three independent experiments] and discharge on the indicated nucleophiles by MALDI-TOF/MS [(C) bars represent the mean of four technical replicates].