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.

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Papillomavirus (PV) E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here we show that recombinant E6 proteins from eight human and one bovine PV strains exist as oligomeric as well as multimeric species. These species were characterized using a variety of biochemical and biophysical techniques including analytical gel filtration, activity assays, SPR, EM and FTIR. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein (MBP), which slows down the formation of higher-order multimeric species. The proportion of each oligomeric form vary depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal HPV E6, retain binding to the ubiquitin ligase E6AP and the capacity to degrade the pro-apoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process which is accompanied by a significant increase in the β-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggest that self-association is a general property of E6 proteins which occurs both in vitro and in vivo and might therefore be functionally relevant.

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognises its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that Bovine Papillomavirus Type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein’s solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein MBP (Maltose-Binding-Protein), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif.