Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain‐16 E2 DNA‐binding domain

Abstract

The equilibrium unfolding reaction of the C-terminal80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, AGHZ0, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, K,,, being 6.5 X lo-' M. Equilibrium sedimentation experiments give a Kd of 3.0 X IO-' M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The K,, changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 X lo-' M to 4.4 x 10" M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10"' M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.Keywords: dimer; E2 DNA-binding domain; equilibrium unfolding; human papillomavirus Many DNA-binding proteins recognize palindromic or partially palindromic DNA sequences. The twofold symmetry of the target DNA sequence is reflected in the proteins, which are often dimeric with a twofold axis of symmetry (Harrison, 1991 Papillomaviruses are small DNA viruses that infect the skin and mucosa of a wide variety of mammals (Howley, 1990). Historically, bovine papillomavirus (BPV) has been the most studied of the papillomaviruses, but the discovery of a link between papillomavirus infection and cervical cancer in humans has led to much recent interest in human papillomaviruses (HPVs), especially the high-risk subtypes such as HPV 16, 18, and 33 (Gissmann, 1992). Papillomavirus E2 protein is a DNA-binding protein that regulates viral transcription and replication (Ham et al., 1991;McBride et al., 1991). The protein recognizes the sequence ACCN,GGT, which occurs several times in papillomavirus genomes. The E2 protein consists of an N-terminal activation domain and a C-terminal DNA-binding domain joined by a linker region rich in proline residues. The protein dimerizes via the DNA-binding domain. In addition to the full-length protein, two smaller species are produced by alternative splicing. The truncated proteins have deletions in the N-terminal domain but have the C-terminal dimerization/DNA-binding domain intact. These molecules can form heterodimers with the full-length E2 protein, and these heterodimers can act as transcriptional re-310

Experimental snapshots of a protein-DNA binding landscape

“…The HPV16 E2C domain folds into a homodimeric β-barrel wrapped by four α helices and two 3 10 helices (19,22,23). The domain is a very stable homodimer with a K d of 0.4 nM under the conditions used in this work (27). Because we perform our measurements at 20 nM E2C or higher concentrations, it is safe to consider the monomer-dimer equilibrium negligible.…”

“…Additionally, other factors may increase the degree of frustration in the landscape in addition to side-chain-DNA contacts, such as the requirement for DNA bending (5,7,19,21) or binding after sliding along nonspecific DNA (3). The free E2C domain is stable (27) yet highly dynamic, with less than half of backbone amide protons showing significant protection from exchange with the solvent (24), while most backbone amide protons are shielded from exchange in DNA-bound E2C (24). Moreover, the conformational diversity of the DNAbinding helix is restricted upon binding (35) and internal conformational rearrangements in the homodimeric β-barrel core take place (24).…”

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

“…Much has appeared in the literature about the behavior of the E2c protein from the high risk HPV-16, in terms of homoassociation (13)(14)(15)(16) and DNA binding and recognition, through equilibrium (6, 9, 14, 16 -20) and kinetic assays (4,21). Although we have previously presented preliminary evidence of a possible equilibrium between free and DNA-bound monomer (16), there is still some doubt about the existence of such an equilibrium.…”

Temperature and Mg2+ Sensing by a Novel PhoP-PhoQ Two-component System for Regulation of Virulence in Edwardsiella tarda

“…The data were curve-fitted according to the equation of Mok et al (31) to obtain the ⌬G values. In the absence of Mg 2ϩ , PhoQs had a higher stability toward urea at 30°C (⌬G ϭ 8.5 kcal/ mol) than at 37°C (⌬G ϭ 7.6 kcal/mol) (Fig.…”