National Center for Bioinformatics, Quaid-i-Azam University, Islamabad, 42000, Pakistan.
National Center for Bioinformatics, Quaid-i-Azam University, Islamabad, 42000, Pakistan.
Comput Biol Med. 2022 Jul;146:105660. doi: 10.1016/j.compbiomed.2022.105660. Epub 2022 May 22.
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 E2 to HECT domains of NEDD4L, WWP1, WWP2, HECW1, and HECW2 through in silico analysis to isolate the E2-specific peptide inhibitors that may target SARS-CoV-2 viral egression. Molecular dynamics analysis revealed more opening of E2-binding pocket upon binding to HECT, HECT, HECT, HECT, and HECT. We observed similar binding pattern for E2 and mentioned HECT domains as previously reported for HECT where Trp762, Trp709, and Trp657 residues of HECT, HECT, and HECT are involved in making contacts with Ser94 residue of E2. Similarly, corresponding to HECT Tyr756 residue, HECT, HECT, HECT, and HECT-specific Phe703, Phe651, Phe1387, and Phe1353 residues execute interaction with E2. Our analysis suggests that corresponding to Cys942 of HECT, Cys890, Cys838, Cys1574, and Cys1540 residues of HECT, HECT, HECT, and HECT, respectively are involved in E2-to-E3 Ub transfer. Furthermore, MM-PBSA free energy calculations revealed favorable energy values for E2-HECT complexes along with the individual residue contributions. Subsequently, two E2-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.
同源的 E6AP 羧基末端（HECT）- 型 E3 连接酶通过与 E2∼Ub 形成复合物来进行泛素（Ub）-蛋白酶体蛋白降解。包括 SARS-CoV-2 在内的包膜病毒通过利用 E 类液泡蛋白分选（ESCRT）机制从受感染的细胞中逃逸，并通过基于 PPAY 基序与 HECT Ub 连接酶活性的连接来模拟细胞系统。在本研究中，我们通过计算机分析鉴定了 E2 与 NEDD4L、WWP1、WWP2、HECW1 和 HECW2 的 HECT 结构域的结合模式，以分离可能针对 SARS-CoV-2 病毒逸出的 E2 特异性肽抑制剂。分子动力学分析表明，E2 与 HECT、HECT、HECT、HECT 和 HECT 结合后，E2 结合口袋的打开程度更大。我们观察到 E2 和提到的 HECT 结构域的结合模式与之前报道的 HECT 中相同，其中 HECT、HECT 和 HECT 的 Trp762、Trp709 和 Trp657 残基与 E2 的 Ser94 残基发生相互作用。同样，对应于 HECT 的 Tyr756 残基，HECT、HECT、HECT 和 HECT 特异性的 Phe703、Phe651、Phe1387 和 Phe1353 残基与 E2 执行相互作用。我们的分析表明，对应于 HECT 的 Cys942，HECT、HECT、HECT 和 HECT 中的 Cys890、Cys838、Cys1574 和 Cys1540 残基分别参与 E2 到 E3 Ub 的转移。此外，MM-PBSA 自由能计算显示 E2-HECT 复合物以及各个残基贡献的有利能量值。随后，测试了两个源自 E2 的肽（His55-Phe69 和 Asn81-Ala96）与 NEDD4L、WWP1、WWP2、HECW1 和 HECW2 的 HECT 结构域的结合能力。通过将 Phe62、Pro65、Ile84 和 Cys85 残基替换为 Ala 验证了它们的结合，这表明结合受到损害，表明所提出的肽配体可能选择性地靶向 E2-HECT 结合和 Ub 转移。总之，我们提出肽驱动的 E2-HECT Ub 加载阻断可能限制 SARS-CoV-2 的逸出和在宿主细胞中的传播。
