SspH2 selectively binds the UbcH5 ∼ Ub conjugate. Overlay of 1H, 15N- TROSY spectra of (A) free 15N-UbcH5 and 15N-Ub (300 μM each) in the absence (Black) and presence (Green) of 300 μM SspH2477–788, (B) 300 μM 15N-UbcH5-O-Ub in the absence (Black) and presence (Red) of 300 μM SspH2477–788, (C) 300 μM 15N-UbcH5-O-Ub in the absence (Black) and presence (Cyan) of 150 μM C580S-SspH2171–788, and (D) 300 μM 15N-A96D-UbcH5-O-Ub in the absence (Black) and presence (Magenta) of 300 μM SspH2477–788. In A and D the overlaid spectra are nearly identical indicating little or no interaction whereas significant perturbations are observed in B and C. Resonances of 15N-labeled UbcH5-O-Ub significantly affected by addition of SspH2477–788 (B) are mapped in red onto a ribbon structure (E) and a surface representation (F) of a model of UbcH5 ∼ Ub based upon the Ubc13 ∼ Ub conjugate (PDB ID 2 gmi). Topological features of UbcH5 described in the text are labeled in E. Residues that correspond to the canonical eukaryotic E3 binding surface located in Helix-1, Loop 4, and Loop 7 and are shown in light pink. The region on UbcH5 where eukaryotic E3 and SspH2 binding surfaces overlap is shown in orange.