2024
Research News
Researchers Reveal Dynamic Regulation of Ubiquitin Ligase E6AP Activity
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E6AP, the founding member of the HECT-type ubiquitin ligase family, is initially discovered for its interaction with the oncogenic protein E6 of human papillomavirus (HPV). E6 from high-risk HPV hijacks the ubiquitin ligase activity of E6AP, leading to abnormal ubiquitination and degradation of the tumor suppressor p53. This process is associated with the development of various HPV-positive cancers, including over 90% of cervical cancers.
HPV E6 not only alters the substrates of E6AP but also enhances its activity through an unknown mechanism. Additionally, deficiencies or enhancements in E6AP activity are associated with Angelman syndrome and autism spectrum disorders, respectively. Therefore, studying the regulatory mechanism of E6AP activity is of great importance.
In a study published in Nature Communications, a team of researchers led by YU Xuekui and LUO Cheng from Shanghai Institute of Materia Medica (SIMM) of the Chinese Academy of Sciences, resolved the structures of E6AP and E6-bound E6AP using cryo-electron microscopy (cryo-EM), and revealed the functional mechanism of E6AP through structural comparison between the E6AP alone and the E6AP/E6 complex, complemented by molecular dynamics simulations and biochemical analysis.
Researchers first found the E6AP/E6 complex exhibits as a homodimer of heterodimer (protomer). Each protomer consists of one E6AP and one E6 molecule. The oncogenic protein E6 binds to a surface pocket, formed by the conserved α1-helix and a loop-helix-loop element of E6AP. The dimerization of E6AP/E6 protomers is primarily mediated by hydrophobic interactions between the carboxy termini of the two conserved α1-helices.
Then, researchers obtained five different conformations of E6AP/E6 complex, and found that the protomers swing around the crossed region of the two α1-helices. The dynamic characters of the E6AP-E6 complex favor the proximity of E6AP’s C-lobe to the ubiquitin-conjugating enzyme E2 for accepting ubiquitin or to substrates for donating ubiquitin. Functional analysis confirmed that the dynamics and dimerization of the E6AP/E6 protomer are required for E6AP effectively transferring ubiquitin to its substrates.
Furthermore, researchers found the α1-helix in E6AP alone exists as a short helix, and the counterpart in the E6AP/E6 complex became a longer helix. Molecular dynamics simulations showed that the extension of the α1-helix is stabilized by E6.
Based on these findings, researchers proposed a dynamic regulatory mechanism of E6AP activity by E6: E6 binding stabilizes the elongation of E6AP α1-helix, thereby promoting the dimerization of E6AP and transforming the inactive E6AP into the active dimer. Actually, some E6AP mutations associated with Angelman syndrome and autism spectrum disorders are located in the extended α1-helix. The effects of these mutations are correlated with the stability or interactability of the extended α1 helix, highlighting the importance of the α1-helix in mediating dimerization interactions.
This study provides crucial structural information for understanding the physiological and pathological mechanisms of E6AP and contributes to the development of therapeutic interventions for diseases associated with E6AP dysfunction.
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