The B-box1 domain of PML mediates SUMO E2-E3 complex formation through an atypical interaction with UBC9.

The small, ubiquitin-like modifier SUMO is covalently attached to substrates by the enzyme UBC9. SUMO conjugation of substrates often requires an E3 ligase, which ensures substrate specificity by simultaneously binding UBC9 and the substrate. E3 SUMO ligases commonly use a RING domain to engage UBC9. The Promyelocytic Leukemia protein (PML) has been implicated as a probable SUMO ligase. Although PML does contain a RING domain, which is expected to recruit UBC9, we demonstrate that PML RING does not bind UBC9 in vitro. Instead, we show that isolated PML B-box1 possesses UBC9-binding activity and map the B-box1 binding site on UBC9. This site also binds the upstream E1 enzyme that transfers SUMO to UBC9. The overlap of these two binding sites suggests that UBC9 cannot interact with its E1 and E3 partners simultaneously. Furthermore, we present a model of the PML dimer that supports the accessibility of B-box1 for UBC9 binding in the context of the full-length PML.