The study investigates the structural mechanisms underlying the functional activity of the ubiquitin ligase E6AP, which is associated with Angelman syndrome and Autism spectrum disorder. E6AP is hijacked by the high-risk HPV E6 protein to aberrantly ubiquitinate the tumor suppressor p53, leading to multiple types of cancer, including cervical cancer. The research reveals that E6AP and the E6AP/E6 complex exist as monomers and dimers, respectively. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6, contributing to dimerization. The extended α1-helices of the dimer intersect symmetrically, facilitating substrate attachment and detachment to the catalytic C-lobe of E6AP, thereby promoting ubiquitin transfer. The dynamic nature of the E6AP/E6 complex, as evidenced by five conformational states, allows the C-lobe to approach E2 for ubiquitin acceptance or to substrates for donation. Site-directed mutagenesis and structural analyses suggest that dimerization of the E6AP/E6 protomer is essential for p53 ubiquitination. The α1-helix plays a crucial role in mediating dimerization and controlling E6AP function, with mutations associated with Angelman syndrome and Autism spectrum disorder affecting its stability and interaction. These findings provide a deeper understanding of the physiological and pathophysiological mechanisms of E6AP function.The study investigates the structural mechanisms underlying the functional activity of the ubiquitin ligase E6AP, which is associated with Angelman syndrome and Autism spectrum disorder. E6AP is hijacked by the high-risk HPV E6 protein to aberrantly ubiquitinate the tumor suppressor p53, leading to multiple types of cancer, including cervical cancer. The research reveals that E6AP and the E6AP/E6 complex exist as monomers and dimers, respectively. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6, contributing to dimerization. The extended α1-helices of the dimer intersect symmetrically, facilitating substrate attachment and detachment to the catalytic C-lobe of E6AP, thereby promoting ubiquitin transfer. The dynamic nature of the E6AP/E6 complex, as evidenced by five conformational states, allows the C-lobe to approach E2 for ubiquitin acceptance or to substrates for donation. Site-directed mutagenesis and structural analyses suggest that dimerization of the E6AP/E6 protomer is essential for p53 ubiquitination. The α1-helix plays a crucial role in mediating dimerization and controlling E6AP function, with mutations associated with Angelman syndrome and Autism spectrum disorder affecting its stability and interaction. These findings provide a deeper understanding of the physiological and pathophysiological mechanisms of E6AP function.