E2UbcH5B-derived peptide ligands target HECT E3-E2 binding site and block the Ub-dependent SARS-CoV-2 egression: A computational study

Homologous to E6AP carboxyl-terminus (HECT)-type E3 ligase performs ubiquitin (Ub)-proteasomal protein degradation via forming a complex with E2∼Ub. Enveloped viruses including SARS-CoV-2 escape from the infected cells by harnessing the E-class vacuolar protein-sorting (ESCRT) machinery and mimic the cellular system through PPAY motif-based linking to HECT Ub ligase activity. In the present study, we have characterized the binding pattern of E2UbcH5B to HECT domains of NEDD4L, WWP1, WWP2, HECW1, and HECW2 through in silico analysis to isolate the E2UbcH5B-specific peptide inhibitors that may target SARS-CoV-2 viral egression. Molecular dynamics analysis revealed more opening of E2UbcH5B-binding pocket upon binding to HECTNEDD4L, HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2. We observed similar binding pattern for E2UbcH5B and mentioned HECT domains as previously reported for HECTNEDD4L where Trp762, Trp709, and Trp657 residues of HECTNEDD4L, HECTWWP1, and HECTWWP2 are involved in making contacts with Ser94 residue of E2UbcH5B. Similarly, corresponding to HECTNEDD4L Tyr756 residue, HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2-specific Phe703, Phe651, Phe1387, and Phe1353 residues execute interaction with E2UbcH5B. Our analysis suggests that corresponding to Cys942 of HECTNEDD4L, Cys890, Cys838, Cys1574, and Cys1540 residues of HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2, respectively are involved in E2-to-E3 Ub transfer. Furthermore, MM-PBSA free energy calculations revealed favorable energy values for E2UbcH5B-HECT complexes along with the individual residue contributions. Subsequently, two E2UbcH5B-derived peptides (His55-Phe69 and Asn81-Ala96) were tested for their binding abilities against HECT domains of NEDD4L, WWP1, WWP2, HECW1, and HECW2. Their binding was validated through substitution of Phe62, Pro65, Ile84, and Cys85 residues into Ala, which revealed an impaired binding, suggesting that the proposed peptide ligands may selectively target E2-HECT binding and Ub-transfer. Collectively, we propose that peptide-driven blocking of E2-to-HECT Ub loading may limit SARS-CoV-2 egression and spread in the host cells.