ABSTRACT
The viral oncoprotein E6 is an essential factor for cervical cancers induced by "high-risk" mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the polyubiquitination of p53 by E6AP.
Subject(s)
Oncogene Proteins, Viral/chemistry , Repressor Proteins/chemistry , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Binding Sites , Escherichia coli , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Magnetic Resonance, Biomolecular , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
Papillomavirus 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 papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the beta-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 suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.