UBC13 and UbcH5 are E2 ubiquitin-conjugating enzymes important in DNA damage response that are inhibited by the deubiquitinase OTUB1. This inhibition is independent of OTUB1's catalytic activity, but requires OTUB1's N-terminal region, which enables OTUB1 to bind preferentially to E2∼ubiquitin conjugates. Two complementary studies now explore the structural mechanism for OTUB1's inhibitory activity. Unexpectedly, both reveal a requirement for an additional unconjugated ubiquitin molecule bound to OTUB1's distal ubiquitin binding site, promoting tighter binding of OTUB1 to E2∼ubiquitin. Wolberger and colleagues present two crystal structures: the C. elegans OTUB1 bound to human UBC13 and the ubiquitin aldehyde (Ubal)-conjugated chimeric C. elegans OTUB1 (with a human N-terminal region) in complex with ubiquitin-conjugated UBC13. The latter reveals that Ubal binding to OTUB1's distal site causes marked structural rearrangements in OTUB1. Modeling shows that the OTUB1 N-terminal helix would clash with UEV1, an ubiquitin E2 variant required by UBC13 to synthesize Lys63-linked chains, thus inhibiting the UEV1-UBC13 interaction and Lys63-linked polyubiquitination. Durocher, Sicheri and colleagues report the crystal structure of human OTUB1 bound to ubiquitin-conjugated UBCH5B, showing OTUB1 engaged with UBCH5B and free ubiquitin. The structure highlights how coupling between OTUB1's proximal and distal ubiquitin-binding sites, critical for deubiquitinase activity, is co-opted for inhibiting E2 enzymes. OTUB1 binding occludes the E3 binding site on UBCH5B and UBC13, providing a general mechanism for the inhibition of E2s. Mutations that abrogate OTUB1's inhibition of ubiquitination in the DNA damage response are located at the interface of OTUB1 with either UBCH5B or ubiquitin; such mutants cannot suppress UBC13 activity in vitro. (Nature doi:10.1038/nature10911, published online 22 February 2012; Mol. Cell 45, 384–397, 2012)