Structural dynamics of the E6AP/UBE3A-E6-p53 enzyme-substrate complex

Abstract

Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 enzyme-substrate complex is assembled, and how E6 stimulates E6AP, is largely missing. Here, we develop and apply different crosslinking mass spectrometry-based approaches to study the E6AP-E6-p53 interplay. We show that binding of E6 induces conformational rearrangements in E6AP, thereby positioning E6 and p53 in the immediate vicinity of the catalytic center of E6AP. Our data provide structural and functional insights into the dynamics of the full-length E6AP-E6-p53 enzyme-substrate complex, demonstrating how E6 can stimulate the ubiquitin ligase activity of E6AP while facilitating ubiquitin transfer from E6AP onto p53.

Multiple regions of E6AP (UBE3A) contribute to interaction with papillomavirus E6 proteins and the activation of ubiquitin ligase activity

“…The E6AP 121-127 region may be required to enable full-length E6AP to outcompete the LQELL peptide in cis with 16E6. As stated previously, XL-MS based modeling of the E6-E6AP interaction proposed that E6AP undergoes a conformational rearrangement upon binding to E6, and that the N and C-terminal regions of E6AP are brought into close proximity [50]. This E6AP conformational rearrangement depends on E6AP residues amino-terminal to amino acids 300.…”

“…It is possible that the proposed conformational orientation of E6-E6AP-p53 requires E6AP amino acids 310-320 and that orienting E6 and the cellular substrate close to the E6AP HECT domain activates E6AP ubiquitin ligase activity. Furthermore, previous studies indicate that E6AP-mediated ubiquitin chain formation relies on the N-terminal region of E6AP [49,50]. If this specifically applies to E6AP residues 310-320, it may be that the processivity of ubiquitin chain formation mediated by E6AP is disrupted with the E6AP 320-875 truncation.…”

“…Indeed, 11E6, which is unable to bind the isolated E6AP LQELL peptide, stimulates E6AP ligase activity to target NHERF1 for degradation, but also requires E6AP residues 310-320 ( Fig 6). A recent study utilized chemical cross-linking coupled to mass spectrometry (XL-MS) to model the E6-E6AP-p53 trimeric complex and postulated that the interaction results in both E6 and p53 being in close proximity to the E6AP active cysteine residue in the HECT domain [50]. It is possible that the proposed conformational orientation of E6-E6AP-p53 requires E6AP amino acids 310-320 and that orienting E6 and the cellular substrate close to the E6AP HECT domain activates E6AP ubiquitin ligase activity.…”

Crystal structure of the catalytic C‐lobe of the HECT‐type ubiquitin ligase E6AP

“…The founding member of the HECT E3 family, E6AP (UBE3A), has key roles in human diseases: its activity is hijacked by the E6 protein from high‐risk human papilloma viruses to promote the proteasomal degradation of the tumor suppressor p53, thereby driving cervical tumorigenesis; genetic up‐regulation of E6AP has been linked to autism spectrum disorders; and the deletion or down‐regulation of this ligase in the brain causes Angelman's syndrome . While the interactions of E6AP with ubiquitin have been analyzed and structural knowledge of its interplay with certain substrates is emerging, no specific inhibitors targeting this crucial ligase are available …”

A synthetic peptide library for benchmarking crosslinking mass spectrometry search engines

“…Chemical crosslinking combined with mass spectrometry (XL-MS) is frequently used to gain structural information on proteins and protein complexes [1,2]. In a crosslinking experiment, a reagent forms covalent bonds between specific amino acid side-chains that are in close spatial proximity, thus revealing distance restraints between residues, and hence interaction sites within a protein or between different proteins [3,4].…”