Natively unfolded proteins.

The papillomavirus E7 proteins.

Structure of the human Papillomavirus E7 oncoprotein and its mechanism for inactivation of the retinoblastoma tumor suppressor.

Structural disorder in viral proteins.

It is recognized now that many functional proteins or their long segments are devoid of stable secondary and/or tertiary structure and exist instead as very dynamic ensembles of conformations. They are known by different names including natively unfolded, intrinsically disordered, intrinsically unstructured, rheomorphic, pliable, and different combinations thereof. Many important functions and activities have been associated with these intrinsically disordered proteins (IDPs), including molecular recognition, signaling, and regulation. It is also believed that disorder of these proteins allows function to be readily modified through phosphorylation, acetylation, ubiquitination, hydroxylation, and proteolysis. Bioinformatics analysis revealed that IDPs comprise a large fraction of different proteomes. Furthermore, it is established that the intrinsic disorder is relatively abundant among cancer-related and other disease-related proteins and IDPs play a number of key roles in oncogenesis. There are more than 100 different types of human papillomaviruses (HPVs), which are the causative agents of benign papillomas/warts, and cofactors in the development of carcinomas of the genital tract, head and neck, and epidermis. With respect to their association with cancer, HPVs are grouped into two classes, known as low (e.g., HPV-6 and HPV-11) and high-risk (e.g., HPV-16 and HPV-18) types. The entire proteome of HPV includes six nonstructural proteins [E1, E2, E4, E5, E6, and E7 (the latter two are known to function as oncoproteins in the high-risk HPVs)] and two structural proteins (L1 and L2). To understand whether intrinsic disorder plays a role in the oncogenic potential of different HPV types, we have performed a detailed bioinformatics analysis of proteomes of high-risk and low-risk HPVs with the major focus on E6 and E7 oncoproteins. The results of this analysis are consistent with the conclusion that high-risk HPVs are characterized by the increased amount of intrinsic disorder in transforming proteins E6 and E7.

Protein intrinsic disorder and human papillomaviruses: increased amount of disorder in E6 and E7 oncoproteins from high risk HPVs.

High-risk papillomaviruses are known to exert their transforming activity mainly through E7, one of their two oncoproteins. Despite its relevance, no structural information has been obtained that could explain the apparent broad binding specificity of E7. Recombinant E7 from HPV-16 purified to near homogeneity showed two species in gel filtration chromatography, one of these corresponding to a dimer with a molecular weight of 22 kDa, determined by multiangle light scattering. The E7 dimer was isolated for characterization and was shown to undergo a substantial conformational transition when changing from pH 7.0 to 5.0, with an increase in helical structure and increased solvent accessibility to hydrophobic surfaces. The protein was resistant to thermal denaturation even in the presence of SDS, and we show that persistent residual structure in the monomer is responsible for its reported anomalous electrophoretic behavior. The dimer also displays a nonglobular hydrodynamic volume based on gel filtration experiments and becomes more globular in the presence of 0.3 M guanidinium chloride, with hydrophobic surfaces becoming accessible to the solvent, as indicated by the large increase in ANS binding. At low protein concentration, dissociation of the globular E7 dimer was observed, preceding the cooperative unfolding of the structured and extended monomer. Although E7 bears properties that resemble natively unfolded polypeptides, its far-UV circular dichroism spectrum, cooperative unfolding, and exposure of ANS binding sites support a folded and extended, as opposed to disordered and fluctuating, conformation. The large increase in solvent accessibility to hydrophobic surfaces upon small pH decrease within physiological range and in mild denaturant concentrations suggests conformational properties that could have evolved to enable protein-protein recognition of the large number of cellular binding partners reported.

High-risk (HPV16) human papillomavirus E7 oncoprotein is highly stable and extended, with conformational transitions that could explain its multiple cellular binding partners.

Despite the fact that E7 is a major transforming oncoprotein in papillomavirus, its structure and precise molecular mechanism of action remain puzzling to date. E7 proteins share sequence homology and proteasome targeting properties of tumor suppressors with adenovirus E1A and SV40 T antigen, two other paradigmatic oncoproteins from DNA tumor viruses. High-risk HPV16 E7, a nonglobular dimer with some properties of intrinsically disordered proteins, is capable of undergoing pH-dependent conformational transitions that expose hydrophobic surfaces to the solvent. We found that treatment with a chelating agent produced a protein that can readily assemble into homogeneous spherical particles with an average molecular mass of 790 kDa and a diameter of 50 nm, as determined from dynamic light scattering and electron microscopy. The protein undergoes a substantial conformational transition from coil to β-sheet structure, with concomitant consolidation of tertiary structure as judged by circular dichroism and fluor...

Peter Gualfetti

Papers

High-risk (HPV16) human papillomavirus E7 oncoprotein is highly stable and extended, with conformational transitions that could explain its multiple cellular binding partners.

The HPV16 E7 viral oncoprotein self-assembles into defined spherical oligomers.