In silico modeling of the cryptic E2~ubiquitin binding site of E6-associated Protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly [Protein Synthesis and Degradation]

To understand the mechanism for assembly of Lys48-linked polyubiquitin degradation signals, we previously demonstrated 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 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 Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys820 prior to 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 Glu550 serves as a general base in generating the Cys820 thiolate within the low dielectric binding interface and that Arg506 functions to orient Glu550 and in stabilizing the incipient anionic transition state during thioester exchange.

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