Positive Contribution of Hydration on DNA Binding by E2c Protein from Papillomavirus

Abstract

Protein-nucleic acid interactions are responsible for the regulation of key biological events such as genomic transcription and recombination and viral replication. However, the recognition mechanisms involved in these processes are not completely understood. Here, we investigate the dominant forces involved in protein-protein and protein-DNA interactions for the 80-amino-acid C-terminal domain of the E2 protein (E2c) from human papillomavirus (HPV-16). The E2c protein is a homodimer that specifically binds to double-stranded DNA containing the consensus sequence ACCG-N 4 -CGGT, where N is any nucleotide. DNA binding affinity is reduced by lowering water chemical potential, accompanied by an increase in cooperativity. Wyman linkage relations between affinity and water chemical potential indicate that 11 additional water molecules are bound in the formation of the complex between E2c and DNA. Salt dissociation isotherms showed that 10 counterions are released upon association, even at low water activity, indicating that this latter variable does not change the electrostatic component of the interaction. Further analysis demonstrates a strong dependence of cooperativity of binding on the protein concentration. Altogether, these results reveal a novel binding pathway in which the consolidated complex may achieve its final form via a monomer-DNA intermediate, which favors the binding of a second monomer. This molecular mechanism reveals the contributions of multiple conformers in a tight virus genome modulation that seems to be important in the cell infection scenario. Human papillomavirus (HPV)1 is a small nonenveloped double-stranded DNA virus that causes epithelial lesions, which often develop into malignancies (1). The viruses HPV 16, -18, and -31 are denoted high risk HPV since they are directly related to cervical cancer. The regulation of gene expression in virus-infected cells is mediated by the factor E2, which controls the transcription of the HPV gene, including those related to malignant transformation. E2 can act either as an activator or as a repressor, depending on the alternate splicing of the product. Infected cells can be found containing (i) the C-terminal domain (E2c), responsible for DNA binding (2) and dimerization (3); (ii) the E2-E8 splicing product; and (iii) the full E2 protein (E2-full), consisting of the E2c, the N-terminal transactivation domain (E2TA), and an unstructured region with little known function. These three components recognize and bind specifically the palindromic sequence ACCGN 4 CGGT (2), where N is any base, although differences in affinity can arise depending on the N bases (4 -7). Part of the E2 structure has been solved: both for E2c, the DNA-binding domain (7-10), and for the transactivation domain (11,12).Much has appeared in the literature about the behavior of the E2c protein from the high risk HPV-16, in terms of homoassociation (13-16) and DNA binding and recognition, through equilibrium (6, 9, 14, 16 -20) and kinetic assays (4, 21). Although we have previously p...

Indirect Readout of DNA Sequence by Papillomavirus E2 Proteins Depends Upon Net Cation Uptake

“…In light of the generally observed displacement of cations upon the binding of proteins to DNA noted above [30][31][32] and the unusual behavior of HPV16-E2/D, 33 . This phenomenon was further investigated using a salt titration assay similar to that used by others to study HPV16-E2/D.…”

“…This phenomenon was further investigated using a salt titration assay similar to that used by others to study HPV16-E2/D. 33 This assay monitors dissociation of protein-DNA complexes as a function of increasing salt concentration. Since this assay is conducted at stoichiometric concentrations of the Figure 5.…”

Identification of a novel ligand binding motif in the transthyretin channel

“…In this case, a 'tight binding' model [45][46][47][48] should apply, since the total amount of ligand significantly differ from free concentration, and thus the total amount of protein is taken into account. For ANS and AmNS binding to TTR, we considered the following two-state reversible equilibrium between these compounds (ligands, 'L') and TTR: TTR þ 2 L½TTR : L 2 Thus, we define the dissociation constant K d of the above reaction according to…”

Investigation of Interactions between DNA and Nuclear Receptors: A Review of the Most Used Methods

“…There are other plots which could be applied to fit anisotropy data; however, it was observed [151][152][153][154] for the majority of NR-DNA interactions that the occupancy of some binding sites may affect the affinity for the unfilled ones. Therefore the Hill approach, which accounts for the possibility that not all receptor sites are independent (cooperativity), may be properly applied in these systems.…”

“…Furthermore, applying Hill approach to fit the anisotropy data, as showed in the following equation, one can determine dissociation constant ( ) and Hill cooperativity coefficient (n) [150,151]:…”