Ordered Self-Assembly Mechanism of a Spherical Oncoprotein Oligomer Triggered by Zinc Removal and Stabilized by an Intrinsically Disordered Domain

Abstract

BackgroundSelf-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism.Methodology/Principal FindingsUsing various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric “Z-nucleus” after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 µM protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a “conformation editing” mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route.ConclusionWe described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions.

Conformational Dissection of a Viral Intrinsically Disordered Domain Involved in Cellular Transformation

“…Addition of TFE stabilized α-helix to different extents in each species, but only E7N presented a pH-dependent increase in helical content at high TFE (Figure 4C). Given the tendency of E7C to oligomerize in the absence of E7N [40], TFE experiments could not be performed on this isolated domain. However, we will assume that the 27–50 fragment cannot stabilize α-helix because i) the highly acidic E7 (25–40) fragment is not sensitive to TFE addition, and ii) the inter-domain “hinge” region is rich in proline residues [29].…”

“…The E7N domain faces the solvent in E7SOs, providing solubility to the otherwise insoluble E7C oligomer [39], [40]. The highly stable E7SOs present amyloid-like properties, display chaperone holdase-like activity [39], [41] and were shown to be located in the cytosol of HPV-transformed cell lines and cancerous tissue, where they can interact with numerous binding partners [25].…”

Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

“…the major oncoprotein of HPV). HPV E7 was indeed shown to be able self-assemble into defined spherical oligomers with amyloid-like properties [64,65], although the possible functional implications of this phenomenon were only discussed in terms of the amyloid-cancer connection [66].…”

A Chlamydomonas-Derived Human Papillomavirus 16 E7 Vaccine Induces Specific Tumor Protection

“…Although the amounts of protein produced are still insufficient for clinical experimentation, the present work shows the possibility of using microalgae for the production of bio-active HPV E7 antigen. Additionally, the obtainment of soluble, purified E7 protein from microalgae offers the possibility to perform detailed biochemical and chemical/physical studies, aimed at verifying its structure and biological activity, which up to now have been performed only on proteins expressed in bacterial systems [45], [46]. Future developments on gene expression optimization (i.e.…”