Biochemical and structural characterization of the ubiquitin-conjugating enzyme UBE2W reveals the formation of a noncovalent homodimer

Abstract

The biochemical and structural characterization of ubiquitin-conjugating enzymes (E2s) over the past 30 years has fostered important insights into ubiquitin transfer mechanisms. Although many of these enzymes share high sequence and structural conservation, their functional roles in the cell are decidedly diverse. Here, we report that the mono-ubiquitinating E2 UBE2W forms a homodimer using two distinct protein surfaces. Dimerization is primarily driven by residues in the ß-sheet region and Loops 4 and 7 of the catalytic domain. Mutation of two residues in the catalytic domain of UBE2W is capable of disrupting UBE2W homodimer formation, however, we find that dimerization of this E2 is not required for its ubiquitin transfer activity. In addition, residues in the C-terminal region, although not compulsory for the dimerization of UBE2W, play an ancillary role in the dimer interface. In all current E2 structures, the C-terminal helix of the UBC domain is at least 15Å away from the primary dimerization surface shown here for UBE2W. This leads to the proposal that the C-terminal region of UBE2W adopts a noncanonical position that places it closer to the UBC ß-sheet, providing the first indication that at least some E2s adopt C-terminal conformations different from the canonical structures observed to date.

Fig. 1

UBE2W exists in a monomer–dimer equilibrium. a Size exclusion elution profiles for UBE2W-WT (blue) and UBE2D3 (red) detect both a monomer and dimer for UBE2W, but only a monomer of UBE2D3. b Mutation of V30 and D67 to lysine in UBE2W (UBE2W-KK) (red) shifts the population of E2 toward a single monomeric peak as compared to UBE2W-WT (blue) c A C-terminal truncation mutant (UBE2W-131D) (red) elutes as both a monomer and dimer, similar to UBE2W-WT (blue). d The ‘KK’ mutation in the UBE2W-131Δ (UBE2W-131Δ-KK) (red) truncation background shifts the E2 population to a single monomeric peak when compared with the UBE2W-131Δ (blue). All size exclusion injections were performed on a column with a relative void volume of −5 mL

Fig. 2

Disruption of the UBE2W dimer does not abrogate its ubiquitin transfer activity. Autoubiquitination of BRCA1 by UBE2W-WT and UBE2W-KK (1 h reaction) is detected in a western blot for FLAG–BRCA1

Fig. 3

Comparison of the NMR spectra of UBE2W-WT and UBE2W-KK. a Full ¹H, ¹⁵N-HSQC spectrum of UBE2W-WT (black) overlayed with the ¹H, ¹⁵N-HSQC spectrum of UBE2W-KK (red). b Region of ¹H, ¹⁵N-HSQC spectrum showing representative resonances shifted between UBE2W-WT (black) and UBE2W-KK (red) (i.e., L25, D40, L55, and L56) and new resonances in the UBE2W-KK spectrum (i.e., F60)

Fig. 4

Surface 1 on UBE2W is involved in crystal contacts. a Domain-swapped UBE2W (truncated at S116) crystal structure (2A7L). The N-terminal helix from one UBE2W subunit (green) noncovalently interacts with the UBC domain of another subunit (cyan), and vice versa. Only one half of the dimer is shown for clarity. b Surface 1 resonances mapped onto the dimerswapped structure (red). In blue are positions V30 and D67, and in yellow is the active site cysteine (C91)

Fig. 5

a Domain architecture and nomenclature of an E2 based on the structure of UBE2D3, the active site C85 is colored in yellow (PDB: 2FUH). b Observed NMR perturbations identify two surfaces on UBE2W. UBE2W homology model based on the structure of UBE2D3 (4DDG) is shown. Positions V30 and D67 (mutated to lysine in the ‘KK’ mutant background) are colored in blue, while red represents both significantly shifted (top 10 % of shifted resonances) resonances between WT and ‘KK’ spectra and new peaks that appear in the ‘KK’ spectrum. Surface 1 includes Loops 4 and 7, the ß-sheet region, and the 3₁₀ helix. Surface 2 is comprised entirely of the C-terminal region. The active site cysteine (C91) is colored yellow. For perturbed residues, only those resonances with unambiguous assignments are colored red

Fig. 6

UBE2W lacking its C-terminal region is stable and retains its ability to form a dimer. Dispersion of resonances in the UBE2W-131Δ spectrum reveals a folded protein