RESUMEN
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. 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.
Asunto(s)
Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Paxillin/química , Dominios y Motivos de Interacción de Proteínas , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Papillomavirus Bovino 1 , Cristalografía por Rayos X , Papillomavirus Humano 16 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/genética , Paxillin/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Estructura Secundaria de Proteína , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes 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 maltose binding protein (MBP), 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.
Asunto(s)
Papillomavirus Bovino 1/genética , Escherichia coli/genética , Proteínas Oncogénicas Virales/genética , Paxillin/genética , Péptidos/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Papillomavirus Bovino 1/metabolismo , Expresión Génica , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/aislamiento & purificación , Proteínas Oncogénicas Virales/metabolismo , Paxillin/aislamiento & purificación , Paxillin/metabolismo , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , SolubilidadRESUMEN
Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.
