In silico modeling of the cryptic E2∼ubiquitin–binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly

Abstract

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.

The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly

“…These latter products were eliminated in the presence of ␤-mercaptoethanol ( Fig. 3, lanes 4 and 5 versus lanes 9 and 10), consistent with assembly of thioester-linked polyubiquitin chains on the Hect domain as has been observed for E6AP (30).…”

“…Finally, we show that Nedd4-2-catalyzed free polyubiquitin chain assembly does not exhibit Ca 2ϩ -dependent C2 domain-mediated autoinhibition, as suggested previously (15,16), but shows competitive inhibition by exogenous C2 domain, which tends to ϳ60% limiting activity. Competitive inhibition suggests the location of the cryptic E2ϳubiquitin-binding Site 1 associated with Hect domain-linked thioester formation that is consistent with in silico modeling recently reported for E6AP (30).…”

Developing Small‐Molecule Inhibitors of HECT‐Type Ubiquitin Ligases for Therapeutic Applications: Challenges and Opportunities

“…This mechanism was recently experimentally validated for WWP1 . On the other hand, kinetic analyses of E6AP and NEDD4‐2 indicate an en bloc/proximal indexation mechanism, in which a ubiquitin chain is preassembled on the ligase active site with the help of two E2 enzymes before the chain is transferred to a substrate . Together, these results are in line with the idea that different ligases follow distinct mechanisms …”

“…A conserved E2‐binding site was shown to reside on the N lobe of the HECT domain, as illustrated by crystal structures of the E2‐bound HECT domains of E6AP and NEDD4‐1 . Recently, the HECT domains of E6AP and NEDD4‐2 were proposed to contain a second E2 binding site, based on kinetic analyses and modeling approaches …”

Semecarpus anacardiumLinn. leaf extract exhibits activities against breast cancer and prolongs the survival of tumor-bearing mice

“…Crystal structures of receptor proteins CDK2 (protein ID: 1W0X) was downloaded from Protein Data Bank (https://www.rcsb.org/structure/2B53), then water molecules and ligand were removed. Patchdock (Tel Aviv University, Israel) was used for molecular docking studies using CDK2 receptor protein and ligands considering settings as clustering RMSD 4.0 and complex type default (Ronchi et al, 2017). The resultant complex of receptor-ligand with solution number, docking score, area and six-dimensional transformation space was recorded.…”