Kinetically driven refolding of the hyperstable EBNA1 origin DNA-binding dimeric beta-barrel domain into amyloid-like spherical oligomers

The Epstein-Barr nuclear antigen 1 (EBNA1) is essential for DNA replication and episome segregation of the viral genome, and participates in other gene regulatory processes of the Epstein-Barr virus in benign and malignant diseases related to this virus. Despite the participation of other regions of the protein in evading immune response, its DNA binding, dimeric beta-barrel domain (residues 452-641) is necessary and sufficient for the main functions. This domain has an unusual topology only shared by another viral origin binding protein (OBP), the E2 DNA binding domain of papillomaviruses. Both the amino acid and DNA target sequences are completely different for these two proteins, indicating a link between fold conservation and function. In this work we investigated the folding and stability of the DNA binding domain of EBNA1 OBP and found it is extremely resistant to chemical, temperature, and pH denaturation. The thiocyanate salt of guanidine is required for obtaining a complete transition to a monomeric unfolded state. The unfolding reaction is extremely slow and shows a marked uncoupling between tertiary and secondary structure, indicating the presence of intermediate species. The Gdm.SCN unfolded protein refolds to fully soluble and spherical oligomeric species of 1.2 MDa molecular weight, with identical fluorescence centre of spectral mass but different intensity and different secondary structure. The refolded spherical oligomers are substantially less stable than the native recombinant dimer. In keeping with the substantial structural rearrangement in the oligomers, the spherical oligomers do not bind DNA, indicating that the DNA binding site is either disrupted or participates in the oligomerization interface. The puzzling extreme stability of a dimeric DNA binding domain from a protein from a human infecting virus in addition to a remarkable kinetically driven folding where all molecules do not return to the most stable original species suggests a co-translational and directional folding of EBNA1 in vivo, possibly assisted by folding accessory proteins. Finally, the oligomers bind Congo red and thioflavin-T, both characteristic of repetitive beta-sheet elements of structure found in amyloids and their soluble precursors. The stable nature of the "kinetically trapped" oligomers suggest their value as models for understanding amyloid intermediates, their toxic nature, and the progress to amyloid fibers in misfolding diseases. The possible role of the EBNA1 spherical oligomers in the virus biology is discussed.     

DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects.

The E1 protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to E1 for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of E1 show that specific basic residues in this region of the protein, which are conserved in all E1 proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of E1 that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.     

Functional interactions between papillomavirus E1 and E2 proteins.

DNA replication of papillomaviruses requires the viral E1 and E2 proteins. These proteins bind cooperatively to the viral origin of replication (ori), which contains binding sites for both proteins, forming an E1-E2-ori complex which is essential for initiation of DNA replication. To map the domains in E2 that are involved in the interaction with E1, we have used chimeric bovine papillomavirus (BPV)/human papillomavirus type 11 (HPV-11) E2 proteins. The results from this study show that both the DNA binding domain and the transactivation domain from BPV E2 independently can interact with BPV E1. However, the roles of these two interactions are different: the interaction between E1 and the activation domain of E2 is necessary and sufficient for cooperativity in binding and for DNA replication; the interaction between E1 and the DNA binding domain of E2 is required only when the binding sites for E1 and E2 are adjacent to each other, and the function of this interaction appears to be to facilitate the interaction between E1 and the transactivation domain of E2. These results indicate that the cooperative binding of E1 and E2 to the BPV ori takes place via a novel two-stage mechanism where one interaction serves as a trigger for the formation of the second, productive, interaction between the two proteins.     

Separate domains in E1 and E2 proteins serve architectural and productive roles for cooperative DNA binding

The E1 and E2 proteins from bovine papillomavirus bind cooperatively to binding sites in the viral origin of DNA replication. The DNA-binding domains (DBDs) of the two proteins interact with each other, and the E2 transactivation domain interacts with the helicase domain of E1. Mutations that disrupt the interaction between the two DBDs also disrupt the interaction between the E2 activation domain and the E1 helicase domain, demonstrating interdependence of the two interactions. Cooperative binding of the two DBDs generates a sharp bend in the DNA that is required for interaction between the E2 activation domain and E1. This indicates that interaction between the two DBDs plays an architectural role, 'triggering' a productive interaction between the E2 transactivation domain and E1 through introduction of a sharp bend in the DNA. This two-step mechanism may be a required feature for cooperative DNA binding to proximal binding sites.     

Identification of the SSB Binding Site on E. coli RecQ Reveals a Conserved Surface for Binding SSB's C Terminus

RecQ DNA helicases act in conjunction with heterologous partner proteins to catalyze DNA metabolic activities, including recombination initiation and stalled replication fork processing. For the prototypical Escherichia coli RecQ protein, direct interaction with single-stranded DNA-binding protein (SSB) stimulates its DNA unwinding activity. Complex formation between RecQ and SSB is mediated by the RecQ winged-helix domain, which binds the nine C-terminal-most residues of SSB, a highly conserved sequence known as the SSB-Ct element. Using nuclear magnetic resonance and mutational analyses, we identify the SSB-Ct binding pocket on E. coli RecQ. The binding site shares a striking electrostatic similarity with the previously identified SSB-Ct binding site on E. coli exonuclease I, although the SSB binding domains in the two proteins are not otherwise related structurally. Substitutions that alter RecQ residues implicated in SSB-Ct binding impair RecQ binding to SSB and SSB/DNA nucleoprotein complexes. These substitutions also diminish SSB-stimulated DNA helicase activity in the variants, although additional biochemical changes in the RecQ variants indicate a role for the winged-helix domain in helicase activity beyond SSB protein binding. Sequence changes in the SSB-Ct element are sufficient to abolish interaction with RecQ in the absence of DNA and to diminish RecQ binding and helicase activity on SSB/DNA substrates. These results support a model in which RecQ has evolved an SSB-Ct binding site on its winged-helix domain as an adaptation that aids its cellular functions on SSB/DNA nucleoprotein substrates.     

Remodeling of the human papillomavirus type 11 replication origin into discrete nucleoprotein particles and looped structures by the E2 protein

The human papillomavirus (HPV) DNA replication origin (ori) shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundreds of base pairs. The HPV type 11 ori spans 103 bp and contains three palindromic E2 binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 ori binding protein. These sites are separated by 64 and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 DNA ori bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound, indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies on the dependence of looping on salt, ATP, and DTT using full-length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N-terminal domain and factors that may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.