ABSTRACT
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 beta-sheet structure, with concomitant consolidation of tertiary structure as judged by circular dichroism and fluorescence. The assembly process is very slow, in agreement with a substantial energy barrier caused by structural rearrangements. The resulting particles are highly stable, cooperatively folded, and capable of binding both Congo Red and thioflavin T, reporters of repetitive beta-sheet structures similar to those found in amyloids, although no fibrillar or insoluble material was observed under our experimental conditions.
Subject(s)
Oncogene Proteins, Viral/chemistry , Papillomaviridae/physiology , Virus Assembly , Benzothiazoles , Casein Kinase II , Circular Dichroism , Congo Red/chemistry , Dimerization , Fluorescent Dyes/chemistry , Humans , Molecular Weight , Oncogene Proteins, Viral/metabolism , Papillomaviridae/chemistry , Papillomavirus E7 Proteins , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Secondary , Solubility , Thiazoles/chemistry , Zinc/chemistryABSTRACT
Anti-double-stranded DNA monoclonal antibodies against a viral transcriptional regulatory site are capable of discriminating single-base replacements with affinities of 1 x 10(-)(9) M, which were optimized for the length of the duplex used as the immunogen. Their affinity for DNA duplexes of increasing length is lower, but reaches a plateau at 2 x 10(-)(8) M, still a fairly high affinity compared to those of most known natural anti-DNA antibodies. The ability of the antibodies to bind to a 166 bp DNA fragment containing the specific sequence strongly suggests that these have the potential of binding the specific sequence within larger genomic DNA fragments. Electrostatic interactions do not play a significant role, the opposite of what is observed in natural DNA binding interfaces. In addition, the insensitivity of the antibody-DNA interaction to solute effects is indicative of a marginal participation of water molecules at the interface compared to the level of participation at the natural E2-DNA interface. Spectroscopic evidence of base unstacking strongly suggests substantial denaturation of antibody-bound DNA, in agreement with thermodynamic results that show an unusual positive heat capacity change, which could be explained at least in part by the exposure of DNA bases upon binding. Lower local DNA stability cooperates with sequence recognition in producing the highest binding affinity. A slow rate of antibody-DNA association indicates an energy barrier imposed by conformational rearrangements, as opposed to an electrostatically assisted diffusion-controlled collision in the E2 DNA binding domain. While the E2-DNA interaction takes place through a typical direct readout mechanism, the anti-double-stranded DNA monoclonal antibody-DNA interaction could be viewed as a distinctive case of indirect readout with a significant distortion in the DNA conformation. However, the precise mechanism with which the DNA bases are accommodated in the antibody combining site will require structural analysis at atomic resolution. These results constitute a first stage for unveiling the unusual molecular recognition mechanism of a specific DNA sequence by antibodies. This mechanism could represent the strategy with which the immune system tightly and specifically recognizes a DNA antigen.
Subject(s)
Antibodies, Antinuclear/metabolism , DNA/immunology , Animals , Antibodies, Monoclonal/metabolism , Antigen-Antibody Reactions , Base Sequence , Binding Sites , DNA/chemistry , DNA/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/immunology , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , In Vitro Techniques , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , Oncogene Proteins, Viral/immunology , Oncogene Proteins, Viral/metabolism , Papillomaviridae/genetics , Papillomaviridae/immunology , ThermodynamicsABSTRACT
The E2 transcriptional activator of the human papillomavirus regulates the expression of most viral transcripts. Its binding to specific target DNA sequences involves large conformational changes in the interacting macromolecules. The high stability of the E2:DNA complex prompted us to analyze the role of macromolecular interactions and adjuvant emulsions in the appearance of conformation-specific antibodies. We demonstrate that immunization with free or DNA-complexed E2 emulsified in an oil-in-water adjuvant elicits a humoral response shifted to the recognition of discontinuous epitopes. Epitope mapping and functional analysis of the generated anti-E2 mAbs reveals that two separate antibodies populations can be obtained: those able to form a stable ternary complex with protein and DNA, and those which recognize the DNA-binding surface of the transcription factor, interfering with E2 binding to DNA.
