The Structural Basis of DNA Target Discrimination by Papillomavirus E2 Proteins
2000; Elsevier BV; Volume: 275; Issue: 40 Linguagem: Inglês
10.1074/jbc.m004541200
ISSN1083-351X
AutoresSeung‐Sup Kim, Jeffrey K. Tam, Ai-Fei Wang, Rashmi S. Hegde,
Tópico(s)Genetic factors in colorectal cancer
ResumoThe 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 (BPV-1) displays no distinctive spacer sequence preference (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 viral genomes reflect these trends; the mucosal HPV genomes have E2 binding sites with AT-rich spacers (Fig.1 a), while the genomes of the nonprimate animal viruses (including BPV-1) have no such predominance of AT-rich spacer-containing binding sites (13Li R. Knight J. Bream G. Stenlund A. Botchan M. Genes Dev. 1989; 3: 510-526Crossref PubMed Scopus (143) Google Scholar). The crystal structures of the E2 DNA-binding domain from strains BPV-1 (14Hegde R.S. Wang A.-F. Kim S.-S. Schapira M. J. Mol. Biol. 1998; 276: 797-808Crossref PubMed Scopus (35) Google Scholar), HPV-16 (15Hegde R.S. Androphy E.J. J. Mol. Biol. 1998; 284: 1479-1489Crossref PubMed Scopus (79) Google Scholar), and HPV-31 (16Bussiere D.E. Kong X. Egan D.A. Walter K. Holzman T.F. Lindh F. Robins T. Giranda V.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 1367-1376Crossref PubMed Google Scholar) have been reported, as has the co-crystal structure of BPV-1 E2 bound to DNA (17Hegde R.S. Grossman S.R. Laimins L.A. Sigler P.B. Nature. 1992; 359: 505-512Crossref PubMed Scopus (325) Google Scholar). However, there is no structural information yet on a DNA complex of any HPV E2 protein. Here we report the crystal structures of the E2 DNA-binding domain (E2/D) from human papillomavirus type 18 and its complexes with high and low affinity binding sites. The HPV-18 E2-DNA interaction uses an induced-fit mechanism, with a large deformation of DNA and only modest local rearrangements of the protein. Comparisons of the various E2/D structures reveal an unexpected similarity between the quaternary structures of the HPV-18 and BPV-1 E2/D proteins as compared with those of the more closely related (in sequence, evolutionary distance of viral strain, and pathology) HPV-16 and HPV-31 E2/D proteins. HPV-18 E2/D binds with very different affinities to the two E2BS sequences used in the crystallographic studies, yet no base sequence-specifying protein-DNA contacts differ in the two complexes. Like the related HPV-16 E2/D protein, but in contrast to BPV-1 E2/D, HPV-18 E2/D recognizes and discriminates against conformational flexibility in DNA. There exists a correlation between the distribution of positive charge on the DNA interaction surfaces of these proteins and their preferences for prebent or flexible DNA targets. His-tagged HPV-18 E2/D (amino acids 286–365 and N-terminal residues GSHM that arose from the cloning procedure) was obtained by overexpression in Escherichia coli strain BL21(DE3)pLysS. The His-tagged protein was purified by nickel affinity chromatography. The His tag was removed by thrombin treatment, and the cleaved protein was further purified by ion exchange chromatography (Fast-S; Amersham Pharmacia Biotech), resulting in pure protein as determined by silver-stained SDS gels. BPV-1 E2/D was purified as described previously (17Hegde R.S. Grossman S.R. Laimins L.A. Sigler P.B. Nature. 1992; 359: 505-512Crossref PubMed Scopus (325) Google Scholar). All oligonucleotides were obtained from the Yale Keck Biotechnology Facility and purified by ion exchange chromatography (Mono-Q; Amersham Pharmacia Biotech) (17Hegde R.S. Grossman S.R. Laimins L.A. Sigler P.B. Nature. 1992; 359: 505-512Crossref PubMed Scopus (325) Google Scholar). All of the crystals were grown by vapor diffusion from hanging drops at room temperature. 5 mg ml−1 of HPV-18 E2/D in 25 mm Tris, pH 7.5, 100 mm NaCl, 10 mm DTT was mixed with an equal volume of a reservoir solution containing 2.7 m ammonium sulfate and 0.1m sodium acetate, pH 4.7. Crystals of dimensions 0.25 × 0.25 × 0.15 mm appeared in 3–5 days. Diffraction data were recorded on a RAXIS II image plate detector from a crystal maintained at −170 °C. An equimolar mixture of HPV-18 E2/D and E2BS(AATT) was made in 25 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm DTT. Drops were made by mixing 10 mg ml−1 complex with an equal volume of a reservoir solution containing 47% 2-methyl-2,4-pentane diol, 100 mm sodium acetate, pH 4.7, 20 mmCaCl2. Platelike crystals of dimensions 0.4 × 0.25 × 0.05 mm appeared in 5–7 days. Diffraction data were recorded on a RAXIS II image plate detector from a crystal maintained at −170 °C. An equimolar mixture of HPV-18 E2/D and E2BS(ACGT) was made in 25 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm DTT. Drops containing equal volumes of the protein-DNA complex and a well solution containing 48% 2-methyl-2,4-pentane diol, 100 mm sodium acetate, pH 4.7, 20 mm CaCl2 were made. Very thin platelike crystals (0.1 × 0.1 × 0.02 mm) appeared in 5–7 days. Diffraction data were recorded at Brookhaven beam X4A on a CCD detector from a crystal maintained at −170 °C. An equimolar mixture of BPV-1 E2/D and an annealed oligonucleotide of sequence CCAACCGAATTCGGTTG was made in 25 mm Tris, pH 7.5, 100 mm NaCl, 10 mm DTT. Drops were made with 6.25 mg ml−1 of the complex and an equal volume of a reservoir solution containing 30% PEG3350, 47 mm MES, pH 6.2, 2 mm CaCl2. Rod-shaped crystals of dimensions 0.15 × 0.1 × 0.08 mm appeared in 5–7 days. Diffraction data were recorded on a RAXIS II image plate detector from a crystal maintained at −170 °C. All images were indexed; the reflections were integrated, scaled, and postrefined with the HKL package (programs DENZO and SCALEPACK (18Otwinowski Z. Sawyer L. Isaacs N. Bailey S. Data Collection and Processing. SERC Daresbury Laboratory, Warrington, UK1993: 56-62Google Scholar)); and the structures were determined by molecular replacement using the program AMORE (19Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5026) Google Scholar). A model of BPV-1 E2/D-E2BS(ACGT) (17Hegde R.S. Grossman S.R. Laimins L.A. Sigler P.B. Nature. 1992; 359: 505-512Crossref PubMed Scopus (325) Google Scholar) was used to find a Molecular Replacement solution for the BPV-1 E2/D-E2BS(AATT) structure. Using data from 15–3.5 Å, the correct solution had a correlation coefficient of 0.25. A polyalanine model constructed from the refined structure of the BPV-1 E2/D-E2BS(AATT) complex was used in Molecular Replacement to determine the structure of the HPV-18 E2/D-E2BS(AATT) complex. Using data from 15–4.8 Å, the correct solution had a correlation coefficient of 0.491. A model consisting of one subunit of HPV-18 E2/D was constructed from the HPV-18 E2/D-E2BS(AATT) complex structure and used to obtain a Molecular Replacement solution for the HPV-18 E2/D structure. Using data between 15 and 4.5 Å, the correct solution had a correlation coefficient of 0.507. Using the refined structure of the E2BS(AATT) complex as a model, rigid body refinement was conducted on the HPV-18 E2/D-E2BS(ACGT) complex data. Using difference maps, the correct oligonucleotide sequence was modeled. All structures (except the HPV-18 E2/D-E2BS(ACGT) complex) were refined using a combination of simulated annealing, bulk solvent correction, positional refinement, and restrained individual B-factor refinement in CNS (20Brunger A. CNS , version 0.9. Yale University Press, New Haven, CT1992Google Scholar). Simulated annealing omit maps were systematically calculated and examined to minimize the effects of model bias. Only restrained grouped B-factors were refined for the HPV-18 E2/D-E2BS(ACGT) structure. The final model of HPV-18 E2/D-E2BS(AATT) includes residues 287–323 and 329–364 (residues 309–312 had weak electron density, and some of these side chains were modeled as Ala). No unambiguous density corresponding to the C-terminal residue (Met365) was present, and it is not included in the refined model. In three out of four subunits that comprise the asymmetric unit, the final model of HPV-18 E2/D includes residues 287–322 and 329–365 (residues 308–312 had weak electron density, and these side chains were modeled as Ala). One subunit had clear density corresponding to residues 323–328, which are included in the model. Since crystal packing influences this region, the loop conformation is not included in any discussions. The final model of HPV-18 E2/D-E2BS(ACGT) includes residues 287–323 and 328–364 (residues 310–312 had weak electron density, and some of these side chains were modeled as Ala). No unambiguous density corresponding to the C-terminal residue (Met365) was present, and it is not included in the refined model. The final model of BPV-1 E2/D-E2BS(AATT) includes residues 326–410. There are two complexes in the asymmetric unit. In one complex, one 5′-overhanging C is flipped out packing against the protein surface, and no density corresponding to the other 5′-C is present. In the other complex, the C nucleotide at position −8 of one strand is flipped out and packed against the protein surface. The 5′-C then base pairs with G+8 of the other strand. Since the DNA conformation at the 5′- and 3′-ends are clearly affected by crystal packing, all analyses of DNA conformation in the discussions are restricted to the central 14 base pairs that include the E2BS. For all structures, the final model had all non-Gly residues in allowed regions of the Ramachandran plot. The data and refinement statistics are summarized in Table I. A section of a composite simulated annealing omit map calculated on the HPV-18 E2/D-E2BS(AATT) co-crystal structure is shown in Fig.2.Table IData collection and refinement statisticsHPV-18E2/DHPV-18E2/D-E2BS(AATT)HPV-18E2/D-E2BS(ACGT)BPV-1E2/D-E2BS(AATT)Resolution range (Å)99–1.999–2.399–3.099–2.25Unique reflections25,00319,322614723,731R merge 1-aR merge: Σh′ 〈‖I h −I h′‖〉 / Σh′ I h′where ‖I h − I h′‖I h − I h′ is the average of the absolute deviation of a reflection from the averageI h of its symmetry mates and Friedel equivalents.7.87.911.38.9Completeness (%)97.588.098.895.Completeness in last shell88.289.299.879.(1.97–1.9)(2.38–2.3)(3.11–3.0)(2.27–2.25)Space groupP212121C2221C2221P212121Unit cella = 41.5a = 69.9a = 71.8a = 52.3b= 46.8b = 82.2b = 82.6b = 54.8c = 161c = 96.1c= 99.1c = 172.7Refinement Resolution range (Å)30–1.930–2.430–3.030–2.3 Reflections used for refinement24,1449277577121,933 Rfactor 1-bR-factor: Σ ‖ ‖F O h‖ − ‖F C ‖ / Σh‖F O h‖, where ‖F O ‖F O is the observed structure factor amplitude and ‖F C ‖ is the structure factor amplitude from the refined coordinates.23.720.023.722.9 FreeR 1-cFree R, R factor calculated on 10% of the data excluded from refinement.29.429.030.226.2 Number of atoms2452184718804081 Number of waters99190082 r.m.s.d. 1-droot mean square deviation. bond lengths (Å)0.0280.0080.0070.006 r.m.s.d. angles (degrees)2.31.31.21.21-a R merge: Σh′ 〈‖I h −I h′‖〉 / Σh′ I h′where ‖I h − I h′‖I h − I h′ is the average of the absolute deviation of a reflection from the averageI h of its symmetry mates and Friedel equivalents.1-b R-factor: Σ ‖ ‖F O h‖ − ‖F C ‖ / Σh‖F O h‖, where ‖F O ‖F O is the observed structure factor amplitude and ‖F C ‖ is the structure factor amplitude from the refined coordinates.1-c Free R, R factor calculated on 10% of the data excluded from refinement.1-d root mean square deviation. Open table in a new tab The oligonucleotides were engineered so that the termini of the DNA were either at one end or within the spacer region (Table II and Fig.3) of the E2 binding site. This strategy allowed the introduction of breaks in the phosphodiester backbone of the E2 binding site. Oligonucleotides were purified by acrylamide gel electrophoresis, end-labeled, and annealed by heating the probe (2 nm concentration) to 90 °C for 10 min and transferring immediately to ice for 10 min. Quantitative gel mobility shift experiments were conducted following published protocols (21Senear D.F. Brenowitz M. J. Biol. Chem. 1991; 266: 13661-13671Abstract Full Text PDF PubMed Google Scholar, 22Senear D.F. Dalma-Weishaus D. Brenowitz M. Electrophoresis. 1993; 14: 704-712Crossref PubMed Scopus (21) Google Scholar). Briefly, serial dilutions of the proteins were added to binding buffer resulting in a final mixture containing 2 nmγ-32P-labeled oligonucleotide, 150 μg/ml bovine serum albumin, 5 μg/ml sonicated salmon sperm DNA, 5.5 mm DTT, 22 mm HEPES, pH 7.9, 150 mm KCl, 5 mm MgCl2, and 10% (v/v) glycerol. The reaction mixtures were incubated in a water bath at 25 °C for 30 min and then loaded onto prerun 12% polyacrylamide gels in 0.5× TBE, pH 8.0. The electrophoresis was run at 200 V for ∼90 min. The gels were dried and the reaction products visualized by exposure to phosphor storage plates, which were scanned using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).Table IIEquilibrium binding constants for HPV-18 E2/DBinding siteK eqK rel 2-aK relrefers to K ACGT/K eq.ΔG(±65%) 2-b±65% refers to the confidence limits.m −1kcal/molE2BS(ACGT)1.8 × 10−71.0−9.2 (.04)E2BS(AATT)1.6 × 10−9112.5−12.0 (.07)E2BS(AAAT)1.2 × 10−815.0−10.8 (.09)E2BS(AAAA)1.7 × 10−810.6−10.6 (.06)E2BS(TTAA)3.3 × 10−85.5−10.2 (.06)Binding site 2-c∥ refers to a break in the sugar-phosphate backbone.K eqK rel 2-dK rel refers toK intact spacer/K eq.ΔG(±65%) 2-b±65% refers to the confidence limits.m −1kcal/molE2BS(AATT)1.6 × 10−91.0−12.0 (.07)E2BS(AA∥TT)2.0 × 10−80.08−10.5 (.08)E2BS(TTAA)3.3 × 10−81.0−10.2 (.06)E2BS(TT∥AA)1.2 × 10−82.7−10.8 (.08)The design of oligonucleotides used in these experiments is shown in Fig. 3. Hairpin oligonucleotides of the sequence shown in (Fig.3 a) were used to determine the affinity of HPV-18 E2/D for E2BS with the spacer sequences indicated in the first section. Dumb-bell shaped oligonucleotides (Fig. 3 b) were used to introduce nicks in the phosphodiester backbone of the DNA probe.2-a K relrefers to K ACGT/K eq.2-b ±65% refers to the confidence limits.2-c ∥ refers to a break in the sugar-phosphate backbone.2-d K rel refers toK intact spacer/K eq. Open table in a new tab The design of oligonucleotides used in these experiments is shown in Fig. 3. Hairpin oligonucleotides of the sequence shown in (Fig.3 a) were used to determine the affinity of HPV-18 E2/D for E2BS with the spacer sequences indicated in the first section. Dumb-bell shaped oligonucleotides (Fig. 3 b) were used to introduce nicks in the phosphodiester backbone of the DNA probe. The density of the electrophoretic band representing the protein-DNA complex was quantitated using the ImageQuant software. Binding isotherms were obtained by monitoring the density of the electrophoretic band representing the protein-DNA complex as a function of protein concentration and analyzed by nonlinear least-squares analysis. The equilibrium binding constant, K, was determined by analysis of the titration curves against the coupled equationsθ1=11+K[X]Equation 1 andθ2=K[X]1+K[X]Equation 2 where Θ1 is the DNA fraction unbound, Θ2 is the DNA fraction complexed with protein,K is the equilibrium association constant, and [X] is the free active protein dimer concentration. The DNA binding activity of the HPV-18 E2/D protein preparations was determined from stoichiometric titrations (22Senear D.F. Dalma-Weishaus D. Brenowitz M. Electrophoresis. 1993; 14: 704-712Crossref PubMed Scopus (21) Google Scholar); the values presented are corrected for this activity. Since the γ-32P-labeled oligonucleotide concentration is much lower than the equilibrium dissociation constants being measured, the approximation that total protein concentration is equal to free protein concentration is made. The standard state Gibbs free energy of binding was calculated from the equilibrium association constant by ΔG 0 = −RT ln K, where R is the gas constant and T is temperature. Each of the values of ΔG 0 reported were determined by the global analysis of at least two independent titrations. The 1.9-Å crystal structure of HPV-18 E2/D and the 2.4-Å structure of its complex with the high affinity binding site E2BS(AATT) are described in detail below. In order to examine the stereochemistry at a low affinity HPV-18 E2/D-DNA interface, the structure of HPV-18 E2/D bound to E2BS(ACGT) has been determined. While the limited resolution (3 Å) of this crystal structure does not permit detailed analyses, it is of sufficient quality to allow comparisons of global features and direct protein-DNA interactions. To provide a direct comparison between the modes of DNA recognition of the HPV-18 and BPV-1 E2/D proteins, the 2.3-Å structure of BPV-1 E2/D bound to E2BS(AATT) has also been determined. This complex is similar in most respects to the previously reported crystal structure of BPV-1 E2/D bound to E2BS(ACGT) (17Hegde R.S. Grossman S.R. Laimins L.A. Sigler P.B. Nature. 1992; 359: 505-512Crossref PubMed Scopus (325) Google Scholar), and only salient features are presented here. In all of the discussions below, comparisons are drawn between the complexes of the HPV-18 or BPV-1 E2/D proteins with the same E2BS sequence, unless explicitly stated otherwise. Each monomer of HPV-18 E2/D folds into an open-faced β-sandwich with a β-α-β-β-α-β topology (Fig. 1 c). One α-helix in each subunit contains all of the amino acid residues involved in half-site recognition and is termed the "recognition helix." Two E2/D monomers associate such that the β-strands at the edges (β2 and β4) form hydrogen bonds with their symmetry mates (Fig.1 c). This results in a continuous eight-stranded antiparallel β-sheet. Buried in the interface are numerous large side chains including Thr287, Ile289, His291, Lys293, Trp320, Trp322, Ile332, Thr334, Thr336, Met363, and Met365 (Fig.4 a). The dimerization interface is extensive, occluding 1625 Å2 of surface area. A herring bone type packing of four Trp residues in the dimer interface is common to all of the mucosal HPV E2 proteins (Fig. 4) and is likely to be a major contributor to the stability of the dimer. As a result of the intricate and closely packed dimer interface, any rearrangement of subunits upon DNA binding would entail a significant energetic penalty. There is a cavity at the center of the barrel, and solvent molecules have been modeled into the electron density clearly apparent in this region. Electron density corresponding to the loop between the recognition helix and β2 (residues 308–312) is weak, indicating local disorder in this region. The loop between strands β2 and β3 is disordered. The E2 proteins from different viral strains differ in quaternary structure (Fig.5 a). The HPV-18 and BPV-1 E2/D proteins are alike in the relative orientation of their subunits while differing from the HPV-16 and HPV-31 E2/D proteins. When one subunit of each protein is superimposed, the nonsuperimposed recognition helices of HPV-16 and HPV-18 E2/D are related by an average translation of 7 Å (Fig. 5 a). Functionally, these features of the E2 proteins are critical, since they dictate the spatial arrangement of side chains presented to the major grooves for DNA sequence recognition. The large variation in subunit orientation among the HPV E2/D proteins suggests either that they undergo unique subunit rearrangements upon DNA binding or that DNA is bent very differently in each of the protein-DNA complexes. E2 dimer architecture is governed by two features: first, the alignment of strands β2 and β4 against their symmetry mates (Fig. 5 b) and, second, the packing of side chains in the barrel core (Fig. 4). While the alignment of the β2strands is invariant, E2 proteins from different viral strains differ in the register of their dyad-related β4 strands (Fig.5 b). When one subunit each of the HPV-18 E2/D and HPV-16 E2/D proteins are superimposed, residues Gly361–Thr364 of HPV-18 E2/D align with residues Gly361–Ser364 of HPV-16 E2/D. The nonsuperimposed subunit is out of register by two residues; Tyr362–Met365 of HPV-18 E2/D lines up with Thr360–Met363 of HPV-16 E2/D. As a result, while the Phe362 residues of HPV-16 E2/D straddle a 2-fold axis, the corresponding Tyr362 residues in HPV-18 E2/D are not symmetrically disposed about a 2-fold axis. In this regard, HPV-18 E2/D resembles BPV-1 E2/D, while the HPV-16 and HPV-31 E2/D proteins are similar to each other (23Liang H. Petros A.M. Meadows R.P. Yoon H.S. Egan D.A. Walter K. Holzman T.F. Robins T. Fesik S.W. Biochemistry. 1996; 35: 2095-2103Crossref PubMed Scopus (59) Google Scholar). It has been proposed that strand register is specified by cross-strand side chain interactions (24Merkel J.S. Sturtevant J.M. Regan L. Struct. Fold Des. 1999; 17: 1333-1343Abstract Full Text Full Text PDF Scopus (91) Google Scholar), and this is clearly evident in the case of these E2/D proteins. The invariant glycine in the β4 strand of all E2 proteins (Gly361 in HPV-18 E2/D and HPV-16 E2/D and Gly403 in BPV-1 E2/D) has
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