Kinetic and equilibrium binding studies of the human papillomavirus type-16 transcription regulatory protein E2 interacting with core enhancer elements

Sanders, Cyril M.; Maitland, Norman J.

doi:10.1093/nar/22.23.4890

Cited by 35 publications

(51 citation statements)

References 45 publications

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“…We have shown that mutations at these positions can significantly reduce the binding of hE2. These results confirm and extend the previous observation that a change from A:T to C:G at this position weakens the binding of the hE2 C-terminal domain (29). In contrast, the consensus bE2-binding site shows no preference for A:T at position 7 and a change from A:T to C:G at this position has been shown to result in a slight increase in the binding of bE2 (8).…”

Section: Discussionsupporting

confidence: 90%

“…As amino acids outside this C-terminal region could form important contacts with the DNA we have used the full-length protein to determine relative dissociation constants for the interaction of hE2 with these sites. The values obtained for the full-length protein are similar to those which have previously been obtained using the isolated C-terminal domain (29). This suggests that, unlike the EBNA1 core domain, the hE2 C-terminal domain is the sole determinant of DNA binding affinity and specificity.…”

Section: Discussionsupporting

confidence: 83%

“…Each flanking domain consists of an ␣-helix and an extended polypeptide chain that form major and minor groove contacts with the DNA (28). Sanders and Maitland (29) have assayed the binding of the isolated hE2 C terminus to the four hE2-binding sites present within the HPV 16 P97 promoter (29). As amino acids outside this C-terminal region could form important contacts with the DNA we have used the full-length protein to determine relative dissociation constants for the interaction of hE2 with these sites.…”

Section: Discussionmentioning

confidence: 99%

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DNA Binding and Bending by the Human Papillomavirus Type 16 E2 Protein

Thain¹,

Webster

Emery³

et al. 1997

Journal of Biological Chemistry

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The human papillomavirus (HPV) 16 E2 protein (hE2) binds to four sites present upstream of the P97 promoter and regulates transcription of the viral E6 and E7 oncogenes. We have determined the relative binding constants for the interaction of the full-length hE2 protein with these sites. Our results show that hE2 binds tightly to site 4, less tightly to sites 1 and 2, and weakly to site 3. Similar results have previously been obtained using a C-terminal fragment of the hE2 protein suggesting that the C-terminal domain is the sole determinant of DNA binding affinity and specificity. Using circular permutation assays we show that binding of the hE2 protein induces the formation of a significant DNA bend and that the hE2-induced DNA bend angle is the same at both tight and weak hE2-binding sites. An alignment of the four hE2-binding sites from the HPV 16 genome suggests that this protein recognizes an extended binding site when compared with the bovine papillomavirus E2 protein. Here we show that the hE2 protein binds tightly to sites containing an A:T or a G:C base pair at position 7 of its binding site but weakly to sites containing either C:G or T:A at this position. Using site-directed mutagenesis we demonstrate that an arginine at position 304 of the hE2 protein is responsible for the recognition of specific base pairs at this position.The recognition of specific DNA sequences by transcription factors is often the first step in the regulation of gene expression. An understanding of how these proteins recognize their target sequences, and the effect that this has on DNA conformation, is central to the question of how genes are controlled. We are studying the human papillomavirus E2 protein, a sequence-specific DNA-binding protein involved in the regulation of viral gene expression and DNA replication. Papillomaviruses infect epithelial cells and induce the formation of benign hyperproliferative lesions or warts. Over 70 distinct types of human papillomavirus (HPV) 1 have been described. Some of these viral types produce lesions that have the potential to undergo malignant transformation. HPV 16 and HPV 18, for example, are thought to play a primary role in the development of cervical cancer (for a review, see Ref. 1). The products of the viral E6 and E7 genes form complexes with the cellular tumor suppressor proteins p53 and Rb, respectively. These interactions bring about a change in cell growth rate and promote cell immortalization (reviewed in Ref.2). In HPV 16, transcription of the E6 and E7 genes is under the control of a single promoter (P97) that lies immediately upstream of the E6 gene (3). The activity of the P97 promoter is regulated by a variety of cellular transcription factors and by the viral E2 protein (4 -6).Much early work concentrated on the bovine papillomavirus (BPV) E2 protein. The BPV E2 protein (bE2) binds as a dimer to 12 inverted repeats (consensus sequence 5Ј-ACCGN 4 CGGT-3Ј) present upstream of the BPV early genes and activates transcription (7,8). Binding of bE2 protein to DNA is coo...

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

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DNA Binding and Bending by the Human Papillomavirus Type 16 E2 Protein

Thain¹,

Webster

Emery³

et al. 1997

Journal of Biological Chemistry

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“…The extremely slow half-time for dissociation (Ͼ4 h, see Ref. 9) and the very high stability of the E2-DNA complex suggests a quasi-irreversible reaction, with strong implications for its mechanism of action. The conditions under which the dissociation rate was measured include a number of additions to the buffer, including 10% glycerol; these may affect the stability and therefore the off-rate.…”

Section: Discussionmentioning

confidence: 99%

“…The protein consists of a C-terminal DNA-binding and dimerization domain (E2-DBD) 1 and an N-terminal transactivation domain, separated by a flexible region (8). The E2-DBD is an ϳ80-residue per monomer domain that can be overexpressed recombinantly in bacteria as a stable and soluble dimer (9,10). Its biological importance is shown by the three forms of the protein produced by alternative splicing: the full-length protein (E2-TA), which acts as a transcriptional activator, and two shorter forms (E2-TR and E2/E8) produced from alternative splicing, acting as repressors (11).…”

mentioning

confidence: 99%

Conformational Changes and Stabilization Induced by Ligand Binding in the DNA-binding Domain of the E2 Protein from Human Papillomavirus

Lima¹,

Prat‐Gay

1997

Journal of Biological Chemistry

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We are investigating the folding of the 81-residue recombinant dimeric DNA binding domain of the E2 protein from human papillomavirus and how it is coupled to the binding of its DNA ligand. Modifications in buffer composition, such as ionic strength and phosphate, cause an ϳ5.0 kcal mol ؊1 stabilization of the domain to urea unfolding, based on very similar conformational changes as measured by far UV circular dichroism. Binding of DNA produces an even greater stabilization, magnitude similar to that caused by the nonspecific polymer ligand heparin, which shifts the urea midpoint 2.5-fold. The DNA-bound complex displays substantial changes similar to those caused by ionic strength and phosphate in terms of overall secondary structure. Bis-8-anilino-1-naphthalenesulfonate provides a very sensitive conformational probe, which shows alterations in the domain caused by the above mentioned compounds. In general terms, binding of DNA involves an overall conformational readjustment in the protein but maintains the ␤-barrel scaffold intact. This conformational plasticity seems to be of importance in the regulatory functions of this type of DNA-binding protein. The extremely long half-life of the E2-DNA complex, together with its very high stability, suggests that, in the absence of other factors that may affect its stability in vivo, the possibility of dissociation once formed is restricted.Molecular interaction between proteins and DNA play a central role in the regulation of gene function in the biological world. DNA-binding proteins interact with specific target sequences in the DNA, and the basis for the recognition process at the molecular level has been the focus of intense research (1). Often, the interaction leads to substantial changes in the conformation of both the nucleic acid and the protein, compared with their free forms, with direct implications for their function (2-4). The formation of a protein-DNA complex yields a new and different thermodynamic and structural entity; both processes, DNA binding and conformational changes in the free forms of both macromolecular partners, are highly coupled. This is more dramatic in the case of dimeric transcription factors with intertwined folding topologies such that their dissociated monomers are unfolded, and the process of folding and association and DNA binding are tightly linked (5-7). Thus, DNA-binding domains may undergo local and global folding processes upon binding to their operator sequences.The E2 transcriptional activator of the human papillomavirus regulates the expression of most viral transcripts and participates in the DNA replication process. The protein consists of a C-terminal DNA-binding and dimerization domain (E2-DBD)1 and an N-terminal transactivation domain, separated by a flexible region (8). The E2-DBD is an ϳ80-residue per monomer domain that can be overexpressed recombinantly in bacteria as a stable and soluble dimer (9, 10). Its biological importance is shown by the three forms of the protein produced by alternative splicing: the full-...

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

1996

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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

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Kinetic and equilibrium binding studies of the human papillomavirus type-16 transcription regulatory protein E2 interacting with core enhancer elements

Cited by 35 publications

References 45 publications

DNA Binding and Bending by the Human Papillomavirus Type 16 E2 Protein

DNA Binding and Bending by the Human Papillomavirus Type 16 E2 Protein

Conformational Changes and Stabilization Induced by Ligand Binding in the DNA-binding Domain of the E2 Protein from Human Papillomavirus

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

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