Modeling of the Cryptic E2∼ubiquitin-binding Site of E6-associated Protein (E6AP)/UBE3A Reveals the Mechanism of Polyubiquitin Chain Assembly

To understand the mechanism for assembly of Lys-linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations. To understand substrate inhibition, we exploited the PatchDock algorithm to model UbcH7∼ubiquitin bound to Site 1, validated by chain assembly kinetics of selected point mutants. The predicted structure buries an extensive solvent-excluded surface bringing the UbcH7∼ubiquitin thioester bond within 6 Å of the Cys nucleophile. Modeling onto the active E6AP trimer suggests that substrate inhibition arises from steric hindrance between Sites 1 and 2 of adjacent subunits. Confirmation that Sites 1 and 2 function in was demonstrated by examining the effect of E6APC820A on wild-type activity and single-turnover pulse-chase kinetics. A cyclic proximal indexation model proposes that Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys before stochastic transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys-linked I-polyubiquitin thioester intermediate. Other studies suggest that Glu serves as a general base to generate the Cys thiolate within the low dielectric binding interface and Arg functions to orient Glu and to stabilize the incipient anionic transition state during thioester exchange.