The activity of many cell regulatory proteins is controlled by covalent modification with ubiquitin. The specificity of the ubiquitin-conjugation system is mediated by the action of E3 ubiquitin-protein ligases. Notably, E3s are frequently deregulated in human diseases including cancer, viral infections, and cardiovascular, immunological as well as neurological disorders. E6AP, which is a member of the family of HECT E3s and encoded by the emUBE3A/em gene, represents a prime example for this notion: Dysregulation or stimulation of E6AP's E3 activity by the E6 oncoprotein of human papillomaviruses (HPVs) decisively contributes to HPV-induced cervical carcinogenesis; inactivation of E6AP by genetic alterations of the emUBE3A/em gene is the cause of the Angelman syndrome, a neurodevelopmental disorder; emUBEA3/em gene amplification resulting in E6AP overexpression is the genetic hallmark of the Dup15q autism spectrum disorder. Thus, a thorough understanding of structure-function relationships of E6AP should both provide intimate insights into the mechanisms, by which interaction partners such as the HPV E6 oncoprotein or missense mutations in the emUBE3A/em gene affect the E3 activity of E6AP, and lay the foundation for the design of small molecules that affect E6AP function and eventually open up new therapeutic strategies. However, our current knowledge about the structure of E6AP is mainly limited to its catalytic HECT domain. Similarly, only little is known about how the E3 activity of E6AP is controlled at the posttranslational level.pThis interdisciplinary project brings together a world-recognized group focusing on E6AP biochemistry and cell biology, structural biologists with a strong record in analyzing structures of E6 oncoproteins and protein-inhibitor complexes, and a mass spectrometry team specialized in deciphering conformational changes and protein-protein interfaces in macromolecular complexes. We will combine X-ray crystallography, cryo-electron microscopy and cross-linking mass spectrometry to elucidate the high-resolution structure of E6AP alone and in complex with interaction partners including the HPV E6 oncoprotein and the cellular protein HERC2, both of which have been shown by us to allosterically stimulate the E3 activity of E6AP. Furthermore, we will characterize the interaction of E6AP with stimulatory small molecules, which were recently identified by us, by biochemical and structural means and investigate whether such small molecule effectors can rescue the activity of E6AP mutants found in individuals with Angelman syndrome. Taken together, the proposed studies will provide intimate insights into the structure of E6AP and how its activity is modulated by interaction with other proteins and with small molecules, eventually paving the way for small molecule strategies in the treatment of E6AP-associated disorders.