The Structural Basis of DNA Target Discrimination by Papillomavirus E2 Proteins

Abstract

The papillomavirus E2 proteins regulate the transcription of all papillomavirus genes and are necessary for viral DNA replication. Disruption of the E2 gene is commonly associated with malignancy in cervical carcinoma, indicating that E2 has a role in regulating tumor progression. Although the E2 proteins from all characterized papillomaviruses bind specifically to the same 12-base pair DNA sequence, the cancer-associated human papillomavirus E2 proteins display a unique ability to detect DNA flexibility and intrinsic curvature. To understand the structural basis for this phenomenon, we have determined the crystal structures of the human papillomavirus-18 E2 DNA-binding domain and its complexes with high and low affinity binding sites. The E2 protein is a dimeric β-barrel and the E2-DNA interaction is accompanied by a large deformation of the DNA as it conforms to the E2 surface. DNA conformation and E2-DNA contacts are similar in both high and low affinity complexes. The differences in affinity correlate with the flexibility of the DNA sequence. Preferences of E2 proteins from different papillomavirus strains for flexible or prebent DNA targets correlate with the distribution of positive charge on their DNA interaction surfaces, suggesting a role for electrostatic forces in the recognition of DNA deformability. The papillomavirus E2 proteins regulate the transcription of all papillomavirus genes and are necessary for viral DNA replication. Disruption of the E2 gene is commonly associated with malignancy in cervical carcinoma, indicating that E2 has a role in regulating tumor progression. Although the E2 proteins from all characterized papillomaviruses bind specifically to the same 12-base pair DNA sequence, the cancer-associated human papillomavirus E2 proteins display a unique ability to detect DNA flexibility and intrinsic curvature. To understand the structural basis for this phenomenon, we have determined the crystal structures of the human papillomavirus-18 E2 DNA-binding domain and its complexes with high and low affinity binding sites. The E2 protein is a dimeric β-barrel and the E2-DNA interaction is accompanied by a large deformation of the DNA as it conforms to the E2 surface. DNA conformation and E2-DNA contacts are similar in both high and low affinity complexes. The differences in affinity correlate with the flexibility of the DNA sequence. Preferences of E2 proteins from different papillomavirus strains for flexible or prebent DNA targets correlate with the distribution of positive charge on their DNA interaction surfaces, suggesting a role for electrostatic forces in the recognition of DNA deformability. E2 binding site human papillomavirus bovine papillomavirus dithiothreitol Networks of hydrogen bonds between amino acid side chains and the functional groups of DNA bases are a well documented mechanism of DNA sequence recognition by proteins. Not as well understood is the stereochemical and energetic basis by which protein-DNA binding affinity is modulated when all of these direct components of interaction are conserved. Biologically, such discriminatory abilities are essential when DNA-binding proteins have to select between multiple binding sites present among a vast excess of nonspecific DNA. This is the case in the papillomaviruses, where the primary transcriptional regulatory protein, E2, is confronted by numerous binding sites on the viral genome (Fig. 1 a). These sites must be occupied in a defined order such that transcription and viral DNA replication proceed in a regulated fashion (1Steger G. Corbach S. J. Virol. 1997; 71: 50-58Crossref PubMed Google Scholar, 2Garrido-Guerrero E. Carrillo E. Guido M. Zamorano R. Garcia-Carranca A. Gariglio P. Arch. Med. Res. 1996; 27: 389-394PubMed Google Scholar, 3Cheng S. Schmidt-Grimminger D.-C. Murant T. Broker T. Chow T.L. Genes Dev. 1995; 9: 2335-2349Crossref PubMed Scopus (290) Google Scholar, 4Rapp B. Pawellek A. Kraetzer F. Schaefer M. May C. Purdie K. Grassmann K. Iftner T. J. Virol. 1997; 71: 6956-6966Crossref PubMed Google Scholar, 5Grassmann K. Rapp B. Maschek H. Petry K.U. Iftner T. J. Virol. 1996; 70: 2339-2349Crossref PubMed Google Scholar, 6DiLorenzo T.P. Chen D. Zhang P. Steinberg B.M. Virology. 1998; 243: 130-139Crossref PubMed Scopus (3) Google Scholar, 7DiLorenzo T.P. Steinberg B.M. J. Virol. 1995; 69: 6865-6872Crossref PubMed Google Scholar).Figure 1a, structure of the HPV-18 genome. The 7857-base pair circular genome consists of two regions coding for early (E) and late (L) genes. An 800-base pair noncoding region is present upstream of the E6 gene and is called the long control region (LCR). The major early promoter (P105) and a differentiation-specific promoter are indicated by arrows. The long control region contains both transcriptional regulatory elements and the origin of replication. A region of the long control region (of HPV strains −18, −16, −11, and −31) that contains the four E2 binding sites (E2BS1–4) is detailedbelow. In each case, the spacer sequence is shown inblue. The inset is the consensus DNA-binding site of the E2 proteins and the sequences of the two binding-sites referred to as E2BS(AATT) and E2BS(ACGT). The identity elements are inred, and the preferred nucleotides are in green. b, amino acid sequence alignments of the HPV-18, HPV-16, and BPV-1 E2 DNA-binding domains. Identical residues are in red, and similar residues are in green. The residue numbering for HPV-18 E2 and BPV-1 E2 are shown as is the secondary structure of HPV-18 E2/D. The arrows represent β-strands, andzigzag lines indicate helices. c, ribbon diagram of the HPV-18 E2 DNA-binding domain.The two subunits (gold and lavender) associate to form an eight-stranded antiparallel β-barrel. Disordered regions are represented bydotted lines. The topology is shownbeside them. The β-barrel is formed by curling thefigure toward the viewer such that the β2strands at the edges can form hydrogen bonds with each other. Theribbon diagram was generated by RIBBONS (42Carson M. J. Mol. Graphics. 1987; 5: 103-106Crossref Scopus (512) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The papillomaviruses are a large family of double-stranded DNA viruses that vary in host species specificity, tissue tropism, and the clinical outcome of infection (8zur Hausen H. de Villiers E.M. Annu. Rev. Microbiol. 1994; 48: 427-447Crossref PubMed Scopus (475) Google Scholar, 9zur Hausen H. Biochim. Biophys. Acta. 1996; 1288: F55-F78Crossref PubMed Scopus (1468) Google Scholar). The E2 protein has the same functions in the over 100 characterized papillomavirus strains: to activate or repress transcription in a context-dependent fashion and to facilitate the initiation of viral DNA replication via interactions with the viral replication protein E1. Differences in the details of transcription and replication control have been documented between the viral strains that infect different host tissues and between the papillomavirus strains that are associated with different conditions in humans such as warts or cervical cancer (2Garrido-Guerrero E. Carrillo E. Guido M. Zamorano R. Garcia-Carranca A. Gariglio P. Arch. Med. Res. 1996; 27: 389-394PubMed Google Scholar, 3Cheng S. Schmidt-Grimminger D.-C. Murant T. Broker T. Chow T.L. Genes Dev. 1995; 9: 2335-2349Crossref PubMed Scopus (290) Google Scholar, 4Rapp B. Pawellek A. Kraetzer F. Schaefer M. May C. Purdie K. Grassmann K. Iftner T. J. Virol. 1997; 71: 6956-6966Crossref PubMed Google Scholar, 5Grassmann K. Rapp B. Maschek H. Petry K.U. Iftner T. J. Virol. 1996; 70: 2339-2349Crossref PubMed Google Scholar, 6DiLorenzo T.P. Chen D. Zhang P. Steinberg B.M. Virology. 1998; 243: 130-139Crossref PubMed Scopus (3) Google Scholar, 7DiLorenzo T.P. Steinberg B.M. J. Virol. 1995; 69: 6865-6872Crossref PubMed Google Scholar). The E2 proteins from all viral strains have in common the fact that they bind a palindromic DNA sequence ACCgNNNNcGGT, referred to as the E2 binding site (E2BS;1 lowercase letters indicate preferred nucleotides, and the NNNN region is called the “spacer”). However, there exist virus strain-specific differences in the abilities of various E2 proteins to discriminate between binding sites. The E2 proteins from the human papillomavirus (HPV) strains that infect mucosa (including the cancer-associated strains HPV-18 and HPV-16 and the wart-causing strain HPV-11) bind with significantly greater affinity to E2BS with spacers rich in AT base pairs (1Steger G. Corbach S. J. Virol. 1997; 71: 50-58Crossref PubMed Google Scholar,10Alexander K.A. Phelps W.C. Biochemistry. 1996; 35: 9864-9872Crossref PubMed Scopus (30) Google Scholar, 11Bedrosian C.L. Bastia D. Virology. 1990; 174: 557-575Crossref PubMed Scopus (42) Google Scholar, 12Hines C.S. Meghoo C. Shetty S. Biburger M. Brenowitz M. Hegde R.S. J. Mol. Biol. 1998; 276: 809-818Crossref PubMed Scopus (64) Google Scholar). On the other hand, the E2 protein from bovine papillomavirus type 1 spacer sequence C.S. Meghoo C. Shetty S. Biburger M. Brenowitz M. Hegde R.S. J. Mol. Biol. 1998; 276: 809-818Crossref PubMed Scopus (64) Google Scholar). The viral these the HPV have E2 binding sites with spacers the of the viruses (including have such of binding sites R. J. G. A. M. Genes Dev. PubMed Scopus Google Scholar). The crystal structures of the E2 DNA-binding domain from strains BPV-1 R.S. M. J. Mol. Biol. 1998; 276: PubMed Scopus Google HPV-16 R.S. J. Mol. Biol. 1998; PubMed Scopus Google and K. F. T. Biol. 1998; PubMed Google have been as has the structure of BPV-1 E2 to DNA R.S. PubMed Scopus Google Scholar). However, there is structural on a DNA of HPV E2 we the crystal structures of the E2 DNA-binding domain from human papillomavirus type and its complexes with high and low affinity binding sites. The HPV-18 E2-DNA interaction an with a large deformation of DNA and of the of the various structures an between the structures of the HPV-18 and BPV-1 proteins as with of the sequence, of viral and HPV-16 and proteins. 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The is of by two of HPV-18 lines with of HPV-16 E2/D. a the residues of HPV-16 a the residues in HPV-18 are a In this HPV-18 BPV-1 the HPV-16 and proteins are similar to each other H. K. T. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). has been that is by side interactions Scopus Google and this is in the case of these proteins. The in the of all E2 proteins in HPV-18 and HPV-16 and in BPV-1 has the to the In HPV-18 the is at with the side of in a hydrogen the In BPV-1 the side of in a of the of and residues an in protein 1998; PubMed Scopus Google Scholar). The side chains of in HPV-18 and in HPV-16 in the formed by the of different proteins are this of BPV-1 E2 has been in an interaction with the that is essential for viral DNA replication G. A. J. Virol. PubMed Scopus Google Scholar). The differences in structure to the of and E2 proteins different viral to with each other M. A. J. Virol. 1997; 71: PubMed Google Scholar). The of the β-barrel is in all E2 proteins. A common of the HPV-16 and is the of an two residues from of HPV-16 such is present in or HPV-18 E2/D. the side in an that residues and (Fig. these differences in both HPV-18 and HPV-16 similar for HPV-18 and HPV-16 of which is DNA of the HPV-18 β-barrel both recognition in major (Fig. a). 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These with to the of the base pair to the spacer region of the E2BS are in DNA for the BPV-1 from from the the between and base pairs at and from the in a DNA and Res. 1998; PubMed Scopus Google Scholar). These with to the of the base pair to the spacer region of the E2BS are in DNA for the BPV-1 from from the the between and base pairs at and from the The complexes formed by the HPV-18 and BPV-1 proteins in two the and the at the The is in the HPV-18 complexes it is in the BPV-1 complexes of spacer sequence. A large of at the of the HPV-18 E2BS(AATT) to this of the of is in the of E2-DNA The between BPV-1 and has a positive in the AT all the other complexes. This in a of the In both E2 proteins a similar deformation of DNA characterized by major and at the interaction surface. The in and are of the E2 protein present in the both HPV-18 and BPV-1 must the same deformation in differences in their for intrinsic DNA must differences in their abilities to the recognition of HPV-18 the of a DNA major and numerous side chains to the edges of the bases a). of the E2 binding site sequence is by a of hydrogen bonds (Fig. with in the BPV-1 (Fig. residues and HPV-18 direct hydrogen bonds with bases at These interactions are the defined by the palindromic DNA sequence. contacts the base at the This interaction to the greater affinity with which HPV-18 to sites that have an AT base pair at this in the HPV-18 genome (1Steger G. Corbach S. J. Virol. 1997; 71: 50-58Crossref PubMed Google (Fig. 1 that an this A. K. D. K. J. Biol. 1997; PubMed Scopus Google Scholar). HPV-18 contacts the of the DNA in both the major and the In direct and contacts have been contacts by and at and of the DNA have in the BPV-1 E2-DNA via residues and all major contacts are between these two complexes. The by is unique to the HPV-18 DNA from the HPV-18 complexes is between and amino in the such as that by the side of BPV-1 E2/D. of the direct protein-DNA contacts are present in the low affinity between HPV-18 and E2BS(ACGT). Although is present at in the where protein-DNA contacts are in the high affinity E2BS(AATT) The affinity of HPV-18 toward a of E2BS that in spacer sequence was These the spacer sequences on the HPV-18 genome and in the The are in HPV-18 with greater affinity to E2BS spacer The between binding affinity and spacer sequence can be as for spacer sequences for HPV-16 and K.A. Phelps W.C. Biochemistry. 1996; 35: 9864-9872Crossref PubMed Scopus (30) Google Scholar, 12Hines C.S. Meghoo C. Shetty S. Biburger M. Brenowitz M. Hegde R.S. J. Mol. 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Biol. 1998; 276: 809-818Crossref PubMed Scopus (64) Google Scholar). differences between the HPV-18 and BPV-1 complexes to this the is as in BPV-1 complexes as it is in the HPV-18 complexes a toward be for the BPV-1 in the of BPV-1 E2 a interaction in the of the DNA. This significantly between groups on the two by The spacer sequence of HPV-18 its to for this electrostatic between the of a In order to the between DNA flexibility and HPV-18 we the affinity of HPV-18 toward E2BS sequences HPV-18 with significantly affinity to a E2BS(AATT) it to the In a in a DNA that is to the E2 DNA-binding the affinity of the interaction is that HPV-18 flexibility in the In a it was shown that HPV-16 binding affinity was when the flexibility of the E2BS was via in the C.S. Meghoo C. Shetty S. Biburger M. Brenowitz M. Hegde R.S. J. Mol. Biol. 1998; 276: 809-818Crossref PubMed Scopus (64) Google Scholar). for the that these HPV E2 proteins discriminate flexibility in their DNA targets from an of DNA S. A. 1998; PubMed Scopus Google which that A (of or A are This with the greater affinity by HPV-18 for E2BS(AATT) for or DNA flexibility can the in DNA or protein it the in from a DNA to a The fact that the HPV interaction is by flexibility in DNA that the is by protein-DNA the BPV-1 E2 protein flexibility in the E2BS C.S. Meghoo C. Shetty S. Biburger M. Brenowitz M. Hegde R.S. J. Mol. Biol. 1998; 276: 809-818Crossref PubMed Scopus (64) Google suggesting that the of these E2 proteins be in the case of the E. interactions P. M. S. A. PubMed Scopus Google Scholar, P. PubMed Scopus Google where a for spacer sequences was the interaction was by DNA between DNA flexibility and protein binding affinity has been is a in the distribution of positive on the DNA of HPV-18 and BPV-1 (Fig. A large of positive charge is present in the region of HPV-18 which is the of the E2BS a). This is to the residues and to the DNA are for sequences they are for This is the electrostatic on and are the for and K. J. Mol. Biol. 1998; PubMed Scopus Google Scholar). E2BS with spacer sequences have that are to the HPV-18 surface. HPV-16 have a similar of positive charge at the of its DNA-binding R.S. J. Mol. Biol. 1998; PubMed Scopus Google it binding sites C.L. Bastia D. Virology. 1990; 174: 557-575Crossref PubMed Scopus (42) Google Scholar, 12Hines C.S. Meghoo C. Shetty S. Biburger M. Brenowitz M. Hegde R.S. J. Mol. Biol. 1998; 276: 809-818Crossref PubMed Scopus (64) Google Scholar). the spacer sequence ability of HPV-16 on electrostatic in the case of HPV-18 E2/D. In to both of these HPV positive charge on BPV-1 is on the E2BS with its to binding sites with spacer and structural are in to the between DNA and the mechanism of DNA replication and gene the of E2 binding sites as the of the E2 protein (1Steger G. Corbach S. J. Virol. 1997; 71: 50-58Crossref PubMed Google Scholar, F. H. J. Virol. 1994; PubMed Google Scholar, F. J. Virol. 1998; PubMed Google Scholar). The have a of four E2BS on their (Fig. 1 a). E2 binding at can specifically viral early gene the of E6 and E2 binding at the sites and to of transcription of the early the E6 and is for viral DNA replication. in that have the viral DNA replication is on host they to activate the transcription of the genes. The E6 and proteins can bind and two of and K. J. PubMed Scopus Google Scholar). However, E6 and protein are associated with A. E. J. 5: PubMed Scopus Google Scholar, A. E. 1987; Scholar, Phelps W.C. J. Virol. 1987; PubMed Google and their to be low for a viral A between and of the early promoter is The order in which the E2 binding sites on the genome are occupied on both the intrinsic affinity of the E2-DNA interaction at each site and the of E2 binding to multiple sites. The HPV-18 E2 protein with the affinity to and with affinity to sites and (1Steger G. Corbach S. J. Virol. 1997; 71: 50-58Crossref PubMed Google Scholar). of this intrinsic affinity by binding to and the order of E2BS as by on the HPV-18 long control region (1Steger G. Corbach S. J. Virol. 1997; 71: 50-58Crossref PubMed Google which all four is that at low of E2, is occupied and is E2 with and E2 to to of transcription and that the E6 and proteins are at a to a S. Schmidt-Grimminger D.-C. Murant T. Broker T. Chow T.L. Genes Dev. 1995; 9: 2335-2349Crossref PubMed Scopus (290) Google Scholar). of DNA to the ability of the E2 proteins to discriminate among consensus E2 binding sites present on the viral the of the E2 proteins are for of gene and replication. and Brenowitz of for and for of the