Fig. 3: VHH1_2G2 binds to the backside region of Ube2G2.

A NMR ¹⁵N-TROSY-HSQC spectrum of ¹⁵N labeled Ube2G2 with VHH1_2G2 (red) overlaid on the spectrum of ¹⁵N labeled (Black) Ube2G2 alone. The estimated combined chemical shifts for individual Ube2G2 residues are shown in ppm (parts per million) values for VHH1_2G2 in the middle panel. Residues that show detectable chemical shifts relative to unliganded Ube2G2 are mapped onto the NMR structure of Ube2G2 (PDB: 2KLY) to the right, with coloring schemes indicating the magnitude (red>orange) of chemical shifts. B NMR ¹⁵N-TROSY-HSQC spectrum of Ube2G2 with the weakly binding VHH0_2G2 (blue); description otherwise similar to (A). C NMR ¹⁵N-TROSY-HSQC spectrum of Ube2G2 with a negative control VHH_SMT3 (green) overlaid on the spectrum of ¹⁵N Ube2G2 spectrum (black). The ¹⁵N-TROSY-HSQC spectrum of Ube2G2 alone (black) at the bottom. D–E Size exclusion chromatography (SEC) profiles of VHH1_2G2 in the presence of (D) C-terminally tagged Ube2G2, and (E) N-terminally tagged Ube2G2. All binding studies were carried out with a 2-fold molar excess of VHH1_2G2 over Ube2G2. SDS-PAGE analysis of samples corresponding to the VHH-E2 complex peaks (red) is shown in the middle panel. SDS PAGE of various ratios of E2:VHH1_2G2 is shown to the right to indicate the expected degree of Coomassie staining for both proteins. Abbreviations: SBP: Streptavidin binding protein, SMT3: suppressor of MIF2 mutations, yeast SUMO protein.