Summary: In ubiquitin-like protein (UBL) cascades, a thioester-linked E2∼UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53''s WHB subdomain. Hrt1 functions like a conventional RING E3, with its N terminus recruiting Cdc53 and C-terminal RING activating Ubc12∼Rub1. Dcn1''s “potentiating neddylation” domain (Dcn1P) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12∼Rub1 discharge and directing Ubc12''s active site to Cdc53. Crystal structures of Dcn1P-Cdc53WHB and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1''s interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12∼Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target. [Copyright &y& Elsevier]