E6 viral oncoproteins are key players in epithelial tumors induced by Papillomaviruses in vertebrates, including cervical cancer in humans. E6 proteins target many host proteins by specifically interacting with acidic LxxLL motifs. Here, we solved the crystal structures of Bovine (BPV1) and Human (HPV16) Papillomavirus E6 proteins bound to LxxLL peptides from the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively. In both E6 proteins, two zinc domains and a linker helix form a basic-hydrophobic pocket, which captures helical LxxLL motifs in a way compatible with other interaction modes. Mutational inactivation of the LxxLL binding pocket disrupts the oncogenic activities of both E6 proteins. This work reveals the structural basis of both the multifunctionality and the oncogenicity of E6 proteins.

). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule. P apillomavirus E6 oncoproteins interact with target cellular proteins through docking on short peptides often containing the sequence LXXLL (4, 6, 26). The cancer-associated human papillomavirus type 16 (HPV-16) protein E6 (16E6) binds to an LXXLL motif (LQELL) on the cellular E3 ubiquitin ligase E6AP (10); 16E6-E6AP recruits and ubiquitinates p53. Neither 16E6 nor E6AP interacts alone with p53 (8-10, 17, 18) (20). A form of E6AP with a C833A mutation still binds E6 and p53 but fails to ubiquitinate or degrade p53 (19).The necessity of E6AP for p53 degradation by 16E6 is controversial. While E6AP is required for in vitro degradation systems (9) and immortalized mouse fibroblasts (5), E6AP-null mice that express 16E6 in the skin do not accumulate p53 when irradiated, and mouse embryo kidney cells from those E6AP-null mice are reported to lose p53 upon overexpression of 16E6 (12,22). Further, ubiquitin-independent degradation of p53 by E6 has been described (2). These disparate observations raise the questions of whether of E6AP is necessary for degradation and how the specificity of 16E6 for p53 is determined.16E6 binding to the LQELL peptide alone recruits p53 to 16E6 in the absence of full-length E6AP. Our previous studies in yeast (Saccharomyces cerevisiae) had shown that p53 was very weakly recruited by LexA fusions to 16E6, but coexpression of LexA_16E6 with the E6AP C833A mutant (here termed E6AP-Ub Ϫ , for ubiquitin ligase negative) strongly recruited p53 to 16E6 (5). We reasoned that the binding of 16E6 solely to the minimal LQELL peptide of E6AP or similar LXXLL peptides might be sufficient to induce a p53-binding conformation of 16E6. To test this, we fused the 10-residue E6AP LQELL peptide between LexA and 16E6 to provide 16E6 with an LQELL docking site in cis; we similarly fused a mutated LXXLL peptide (LQEAS) between LexA and 16E6 (Fig. 1A). We tested interactions of these LexA fusions by yeast two-hybrid assays with a B42 transactivator-tagged LQELL peptide, B42_E6AP-Ub Ϫ , or with B42_E6AP-Ub Ϫ molecules where LQELL was progressively mutated from LQELL to LQELS or LQEAS. Fusion of the LQELL peptide in cis to 16E6 blocked interaction with B42_LQELL peptide in trans as expected, and mutation of LexA_LQELL_16E6 to LexA_LQEAS_16E6 restored interactions in trans (Fig. 1B, spots 4C and 4D). This is consistent with the LQELL peptide binding in cis to the 16E6 portion of the fusion protein and blocking trans interaction with the B42-fused peptide. Interaction of LQELL_16E6 with B42_E6AP-Ub Ϫ in trans was not blocked, indicating that the interaction of 16E6 with the full E6AP protein is more robust than the interaction with the free LQELL peptide but is still dependent upon an in...