In silico 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 Lys⁴⁸-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 Cys⁸²⁰∼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 in silico 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 Cys⁸²⁰ 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 trans 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 Cys⁸²⁰ before stochastic en bloc transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys⁸²⁰-linked ¹²⁵I-polyubiquitin thioester intermediate. Other studies suggest that Glu⁵⁵⁰ serves as a general base to generate the Cys⁸²⁰ thiolate within the low dielectric binding interface and Arg⁵⁰⁶ functions to orient Glu⁵⁵⁰ and to stabilize the incipient anionic transition state during thioester exchange.

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