Structure of a RING E3 Trapped in Action Reveals Ligation Mechanism for the Ubiquitin-like Protein NEDD8

Abstract

SUMMARY Most E3 ligases use a RING domain to activate a thioester-linked E2~ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2~UBL-target intermediate representing RBX1-UBC12~NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2~UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RINGE2~UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.

Structural mechanisms of HECT-type ubiquitin ligases

“…A similar hydrophobic tethering mechanism for the donor ubiquitin (or donor Ubl) promotes catalytic efficiency and processivity of E2 enzymes (Stewart et al, 2016) and was shown to be stabilized by RING-type E3 enzymes (Dou et al, 2012(Dou et al, , 2013Plechanovová et al, 2012;Pruneda et al, 2012;Scott et al, 2014;Branigan et al, 2015;Wright et al, 2016) as well as SUMO-ligases (Reverter and Lima, 2005;Cappadocia et al, 2015;Streich and Lima, 2016). Importantly, however, the interactions of HECT E3 or E2 enzymes with the donor ubiquitin do not determine the linkage specificity of ubiquitin chain formation.…”

Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation

“…On binding a charged E2, the RING domain stabilizes a closed conformation between the E2 and its donor ubiquitin (14,(27)(28)(29)(30). Once this ubiquitin is transferred to a target lysine, the E2 dissociates from the RING domain to allow for its recharging by the E1 (31).…”

Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme

“…During the modeling efforts, a structure of Rbx1 in complex with Ubc12 oxyesterified to Nedd8 was published (PDB ID 4P5O [7]), and Rbx1 from this cocrystal was therefore used to regenerate the Ube2R1-Rbx1 complex followed by more-extensive modeling of the Ube2R1 acidic loop. Based on the results from the chargeswapped mutations between Arg 91 on Rbx1 and Asp 102 on Ube2R1, ambiguous constraints were implemented between Arg 91's three guanidinium nitrogen atoms and Asp 102's two carboxyl oxygen atoms.…”