©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Overexpression, Purification, and Crystallization of the DNA Binding and Dimerization Domains of the Epstein-Barr Virus Nuclear Antigen 1 (*)

(Received for publication, May 12, 1995 )

Jean A. Barwell (1)(§) Alexey Bochkarev (1) Richard A. Pfuetzner (1) Harry Tong (1) Daniel S. C. Yang (2) Lori Frappier (1)(¶)(**) Aled M. Edwards (1)(¶)(§§)

From the  (1)Institute for Molecular Biology and Biotechnology, Cancer Research Group, and the (2)Department of Biochemistry, McMaster University, 1200 Main Street W., Hamilton, Ontario L8S 4B2, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The Epstein-Barr virus nuclear antigen (EBNA) 1 binds to and activates DNA replication from the latent origin of Epstein-Barr virus. Six different fragments of EBNA1 that retain DNA binding activity were expressed in bacteria, purified, and crystallized. Two fragments, EBNA and EBNA, formed well ordered crystals that diffracted beyond 2.5-Å resolution. Two different EBNA crystals were grown from sodium formate, pH 6-6.5. One crystal belonged to the trigonal space group P3 with unit cell dimensions a = b = 86.5 Å and c = 31.8 Å and with two molecules in the asymmetric unit. The other crystal, which appeared only twice and was likely related to the P3 crystal form, belonged to the trigonal space group P312 with cell dimensions a = b = 86.7 Å and c = 31.8 Å. Crystals of EBNA were grown by lowering the salt concentration to 0-100 mM NaCl at pH 6.0. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and had cell dimensions a = 59 Å, b = 66.9 Å, and c = 69.8 Å with two molecules in the asymmetric unit.


INTRODUCTION

DNA replication initiates at discrete points in the genome, termed origins of replication. Origin DNA sequences are recognized by specialized origin DNA-binding proteins (OBPs) (^1)that serve as assembly sites for the DNA replication machinery and can also mediate the local unwinding of origin DNA (reviewed in Kornberg and Baker(1992)). OBPs have been purified and characterized from many different systems including bacteria, plasmids, phages, viruses of mammalian cells, and the yeast Saccharomyces cerevisiae. The OBPs are distinguished by several features: they form multimeric complexes at their respective origins, they lack sequence homology to other characterized DNA-binding proteins, and they are involved in a network of protein-protein interactions with the other components of the replication machinery (Kornberg and Baker, 1992; Kelman and O'Donnell, 1994; Stillman, 1994). The structural mechanisms by which OBPs bind origin DNA and facilitate the assembly of the replication apparatus are unclear. Some insights will undoubtedly arise from the high resolution structure of an OBP and of an OBP bound to its cognate recognition sequence. As a first step toward this goal, we have crystallized the DNA binding domain of an OBP, the Epstein-Barr virus nuclear antigen 1 (EBNA1).

EBNA1 binds as a dimer to multiple copies of an 18-base pair palindromic sequence present within the latent origin of replication of the Epstein-Barr virus (Rawlins et al., 1985; Frappier and O'Donnell, 1991; Ambinder et al., 1991). During latent infection of host cells, Epstein-Barr virus episomal genomes replicate autonomously, initiating bidirectional replication from the latent origin once every cellular S phase (Adams, 1987; Yates and Guan, 1991). This replication is absolutely dependent on EBNA1 and presumably also requires the cellular replication apparatus (Yates et al., 1985). The mechanism by which EBNA1 activates replication from the latent origin is not yet clear because this protein does not appear to have any intrinsic enzymatic activities (Frappier and O'Donnell, 1991; Middleton and Sugden, 1992). In addition to its role in DNA replication, EBNA1 has been shown to activate transcription (Reisman and Sugden, 1986; Sugden and Warren, 1989), repress transcription (Sample et al., 1992), and govern the stable segregation of Epstein-Barr virus episomes during cell division (Krysan et al., 1989). Like the DNA replication function of EBNA1, all of these functions require the binding of EBNA1 dimers to its DNA recognition sequence.

One approach we have taken to better understand EBNA1 function involves the structural analysis of the protein. To begin with we have focussed on the DNA binding and dimerization region, because this region is central to all known EBNA1 functions. Our strategy for structure solution was to purify and crystallize the DNA binding domain of EBNA1 for analysis by x-ray diffraction. The DNA binding and dimerization region of EBNA1 had been previously mapped to the COOH-terminal third of the protein within amino acids 459-607 (Chen et al., 1993). We chose to refine the borders of the domain by limited proteolytic digestion, re-clone the newly defined DNA binding domain for expression in bacteria, and then purify that domain for crystal trials.


MATERIALS AND METHODS

Cloning of EBNA1 DNA Binding and Dimerization Domains into Bacterial Expression Vectors

Six mutants of EBNA1, all containing the DNA binding/dimerization region, were generated. These are EBNA, EBNA, EBNA, EBNA, EBNA, and EBNA, where the subscripts indicate the amino acid coordinates within the protein (see Fig. 1). The coding sequence for each mutant was amplified from the EBNA1 gene in plasmid p205 (Yates et al., 1985) using Vent polymerase (New England Biolabs) by polymerase chain reaction. The restriction sites NdeI and BamHI were added to the 5` and 3` ends of the polymerase chain reaction product, respectively, and were used to insert the EBNA1 fragments between the NdeI and BamHI sites of the T7 polymerase expression vector, pET15b (Novagen). The proteins were thus expressed as COOH-terminal fusions to an NH(2)-terminal six-histidine tag and a thrombin protease site. Digestion of each fusion protein with thrombin resulted in the inclusion of four residues (GSHM) at the NH(2) terminus of the EBNA1 derivatives. The proteins were expressed in Escherichia coli (BL21(DE3)pLysS) (Studier et al., 1990). The polymerase chain reaction primers were designed to yield amplification products with an NdeI site at the 5` end and a BamHI site at the 3` end and are as follows (written 5` to 3`): EBNA: 5` primer CGTCGACATATGGGTCAGGGTGATGGAGGC and 3` primer CGTGCAGGATCCTCACTCCTGCCCTTCCTCACC; EBNA: 5` primer CGTCGACATATGCGCAAAAAAGGAGGGTGG and 3` primer CGTGCAGGATAATCACTCCGCGGCAGCCCCTTCCAC; EBNA: 5` primer CCTCCACATATGGGTCAAGGAGGTTCCAAC and 3` primer CGTGCAGGATAATCACTCCGCGGCAGCCCCTTCCAC; EBNA 5` primer CGTCGACATATGCGCAAAAAACCACCCTGG and 3` primer CGTCGAGGATCCTCAAGGCAAATCTACTCCATC; EBNA: 5` primer CCTCCACATATGGGTCAAGGAGGTTCCAAC and 3` primer CGTGCAGGATCCTCAAGGCAAATCTACTCCATC; and EBNA: 5` primer CGTCGACATATGCATCGTGGTCAAGGAGGT and 3` primer CGTGCAGGATCCTCAAGGCAAATCTACTCCATC.


Figure 1: The EBNA1 truncation mutants. The EBNA1 amino acids present in each truncation mutant and some of the salient features of EBNA1 are shown.



Expression and Purification of Recombinant Proteins

Bacterial cells expressing EBNA were grown in a fermenter (30 liters) in Terrific broth at 37 °C to an absorbance of 0.8 at 595 nm, and EBNA1 expression was induced with 0.5 mM isopropyl beta-D-thiogalactopyranoside, followed 30 min later by the addition of 150 µg/ml of rifampicin. 3 h postinduction, the cells were harvested by centrifugation and resuspended in 20 mM Tris, pH 7.5, 10% sucrose, 1.0 mM benzamidine, 1.0 mM phenylmethylsulfonyl fluoride, and 1.0 mM EDTA (1 ml/gram of cells). Cells were frozen at -70 °C and then thawed and lysed by sonication. NaCl was added to 350 mM, and after a 30-min incubation on ice, the lysate was clarified by centrifugation at 20,000 g for 30 min. The supernatant was then placed in a 75 °C water bath (for EBNA) or in a 55 °C water bath (for the other mutants) until the temperature of the protein solution reached the temperature of the bath. Heating was followed by a 10-min incubation on ice. The supernatant was clarified by centrifugation at 20,000 g for 30 min and then flowed through a 60-ml DE52 column (Whatman) equilibrated with 20 mM Tris, pH 7.5, 350 mM NaCl, 10 mM dithiothreitol, and 10% glycerol. The DE52 flow-through was diluted with 50 mM Hepes, pH 7.2, to a final NaCl concentration of 200 mM and loaded on to a 120-ml heparin-agarose column (Bio-Rad) equilibrated with buffer A (50 mM Hepes, pH 7.2, 200 mM NaCl, 10% glycerol, and 10 mM dithiothreitol). After washing the column with four column volumes of buffer A, the protein was eluted with buffer A containing 750 mM NaCl. The protein eluate was dialyzed against buffer B (50 mM Hepes, pH 7.2, 750 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine) and loaded onto a 5-ml high pressure liquid chromatography metal-chelating column (Perseptive Biosystems) charged with nickel and equilibrated in buffer B plus 5 mM imidazole. The column was developed with a 20-ml linear gradient of 5-300 mM imidazole in buffer B. The fractions containing >99% pure EBNA1 (as judged by Coomassie staining of 15% SDS-polyacrylamide gels) were pooled and digested with thrombin (10 units/mg of protein) at 37 °C for 2.5 h in 50 mM Hepes, pH 7.2, 500 mM NaCl, 10 mM dithiothreitol, 10% glycerol, and 2.5 mM CaCl(2). After digestion, the protein was diluted to 1 mg/ml with the digestion buffer and then diluted further with buffer A lacking NaCl to a final NaCl concentration of 200 mM. This dilution protocol was necessary to avoid protein aggregation. The protein was loaded onto a 5-ml high pressure liquid chromatography Poros S column (Perseptive Biosystems) at 200 mM NaCl in buffer A eluted with a 10-ml linear gradient from 200 mM to 1 M NaCl in buffer A, concentrated to 10 mg/ml using a Centricon-10 microconcentrator (Amicon), and finally dialyzed into buffer C (1 mM Hepes, pH 7.2, 10 mM dithiothreitol, and 500 mM NaCl).

Crystallization of EBNA1

EBNA EBNA, EBNA, and EBNA were crystallized using the method of hanging drop vapor diffusion using Linbro multiwell tissue culture plates. Unless otherwise stated, the crystals were grown at room temperature from an initial protein solution of 10 mg/ml, and the hanging drop comprised an equal volume of the protein solution and the solution containing the precipitant. All solutions, including the protein, were filtered through 0.22 µM filters before crystallization.


RESULTS AND DISCUSSION

The initial goal of this work was to identify the smallest fragment of EBNA1 that harbored DNA binding activity. To accomplish this, EBNA was subjected to limited protease digestion. Two slightly smaller, yet active, trypsin-resistant fragments were found; NH(2)-terminal sequencing revealed that the major peptide began at amino acid 470 with a minor component beginning at amino acid 468. Electrospray mass spectroscopy indicated that the mass difference between EBNA and the proteolytic products was due only to NH(2)-terminal truncations. Resistance to trypsin digestion suggested that these EBNA fragments were more tightly folded and therefore good candidates for crystallization. These studies pointed to amino acid 470 or 468 as the approximate NH(2)-terminal boundary of the DNA binding domain. The COOH-terminal boundary could not be as well defined in these studies due to the lack of suitable protease sites near amino acid 619 and therefore was selected on the basis of previous studies that indicated that the DNA binding/dimerization region extended only to amino acid 607 (Ambinder et al., 1991). We therefore generated and purified the truncation mutants EBNA, EBNA, and EBNA (Fig. 1). Purification of these derivatives was as described above for EBNA, except, to maintain solubility, the salt concentration was maintained at 750 mM NaCl during the digestion with thrombin and while concentrating to 10 mg/ml in buffer C. EBNA was concentrated in buffer C containing 1 M NaCl. All of these EBNA1 fragments were shown by electrophoretic mobility shift assays and methylation protection footprints to bind specifically to the 18-base pair EBNA1 recognition sequence. (^2)The purified proteins are shown in Fig. 2.


Figure 2: SDS-polyacrylamide gel of the EBNA1 truncation mutants. 1.0 µg of protein purified from E. coli lysates was subjected to electrophoresis on a 15% polyacrylamide gel containing SDS and stained with Coomassie Blue. Molecular masses in kilodaltons are shown to the left of the gel. The faint higher molecular mass bands visible in the last four lanes are nondenatured dimers of EBNA1.



Each purified protein was subjected to crystallization trials. To crystallize EBNA, the protein was mixed with an equal volume of 50 mM MES, pH 6.0, and 0.1 M ammonium phosphate and incubated over a reservoir containing the same solution. Hexagonal plates with dimensions 0.1 0.1 0.02 mm or rectangular rods with dimensions 0.05 0.05 0.01 mm grew in 1-7 days. Smaller hexagonal plates could also be induced to grow from stearylamine/sphingosine lipid layers on the coverslip (Hemming et al., 1995). We were unable to generate EBNA crystals that were large enough for x-ray diffraction.

Several crystal forms of EBNA were grown. Hexagonal crystals (0.2 0.2 0.2 mm) grew at room temperature in 4-6 weeks over a reservoir containing 0.5 ml of a solution containing 50 mM MES, pH 6.5, and 0.7-0.9 M NaAc. Another crystal form (0.25 0.05 0.05-mm rectangular rods) grew over a reservoir of 50 mM Hepes, pH 7.0-7.5, 7-10% polyethylene glycol 4000, and 500 mM NaCl. Diffraction analysis revealed that these crystal forms were twinned and not ideal for x-ray analysis.

Two other crystal forms of EBNA grew in 4-6 weeks over a reservoir solution containing 50 mM MES, pH 6-6.5, and 0.8-1.0 M sodium formate. Both crystals had a hexagonal morphology and grew to a maximal dimension of 0.2 0.2 0.2 mm. Diffraction from these crystals was measured at room temperature on an RAXIS II image plate area detector with CuKalpha radiation from a Rigaku RU200 rotating anode generator. The x-rays were focussed to a 0.2-mm spot with Supper double focussing mirrors. The diffraction from each crystal extended to beyond 2.5 Å resolution. These two crystal forms could only be distinguished by diffraction analysis. The first crystal was trigonal with a unit cell of a = b = 86.7 Å and c = 31.8 Å, and the space group assigned as P312. Only two crystals with this space group were detected and in these instances co-existed in the same drop with another crystal form, which was also trigonal with a very similar unit cell of dimensions (a = b = 86.5 Å and c = 31.8 Å). The space group of the second crystal form was tentatively assigned as P3. In the P3 crystal, the calculated unit-cell volume was 205,106 Å^3, and using a protein density of 1.3 g/cm^3, one can infer two molecules in the asymmetric unit and a solvent content of 39%. A heavy atom derivative of the P3 crystals has been prepared by soaking the crystals in mercury acetate, and the space group assignment was confirmed. A self-rotation function showed several possible positions for the noncrystallographic axis (Fig. 3). Of these possibilities, the most probable position of the noncrystallographic axis is that parallel to the ab plane (as indicated) because the noncrystallographic axis in the P3 crystal is probably near to the crystallographic 2-fold in the very closely related P312 crystal form. A native data set was collected from the P3 crystals, and the statistics are shown in Table 1.


Figure 3: Self-rotation function of the P3 crystal showing the several possible positions for the noncrystallographic axis. The self-rotation analysis was performed with 15-5-Å resolution data using MERLOT (Fitzgerald, 1988). C(min) = 72.80; C(max) = 100.00; Delta = 2.08; kappa = 180.





Crystals of the EBNA and EBNA fragments were grown over a reservoir solution containing 50 mM MES, pH 6.0, and 0-100 mM NaCl. Rectangular rods with dimensions of 0.5-1.0 0.2 0.2 mm grew at room temperature in 2-3 days. X-ray diffraction analysis showed that these crystals were tetragonal with unit cell dimensions a = 59 Å, b = 66 Å, and c = 69 Å; the space group assignment was P2(1)2(1)2(1). The diffraction extended to beyond 2 Å, and a heavy atom derivative was obtained by soaking in a solution containing para-chloromercurobenzoate. The interpretation of the Patterson maps permitted the location of several heavy atom sites as well as the confirmation of the space group assignment. Similar analysis to that described above for the P3 crystals indicated that there are two molecules in the asymmetric unit of the P2(1)2(1)2(1) crystal and that the solvent content was 45%. A self-rotation function showed several possible directions for the noncrystallographic 2-fold axis (Fig. 4). At present, we have not been able to determine which is the correct location of the NC axis.


Figure 4: Self-rotation function of the P2(1)2(1)2(1) crystal showing the several possible positions for the noncrystallographic axis. The self-rotation analysis was performed with 15-5-Å resolution data using MERLOT (Fitzgerald, 1988). C(min) = 7.34; C(max) = 100.00; Delta = 3.67; kappa = 180.



Native data sets were collected from the P2(1)2(1)2(1) crystals both at room temperature and at -175 °C. The low temperature was maintained using a nitrogen gas delivery system from Molecular Structure Corporation. The crystals were cryoprotected by sequential 5-min incubations of the crystal in the mother liquor appended with 5, 10, 15, and 20% glycerol, mounted in a loop made from a strand of dental floss, and then flash frozen in the stream of nitrogen gas. The statistics for both room temperature and frozen data sets are shown in Table 1.

To date, DNA binding proteins that activate DNA replication from origin sequences have been recalcitrant to crystallization. For EBNA1, we used a combination of deletion analysis and partial proteolysis to delineate a set of soluble protein fragments that retains the activity of interest and then subjected each to crystallization trials. Each soluble, proteolytically stable domain formed crystals quite readily, attesting to the utility of this approach. However, in our studies, only two of the five EBNA1 fragments that we crystallized yielded crystals suitable for structure determination. These data suggest that stability to protease digestion should be used only as the first approximation of a suitable domain for crystallization and that the most suitable fragment must be identified by testing a set of proteins that contain amino and carboxyl termini that either flank or impinge on the proteolytically defined domain. Diffraction quality crystals will likely be grown from within this set of soluble domains.


FOOTNOTES

*
This work was initiated at the Cold Spring Harbor course on x-ray crystallography and was supported by grants from the National Cancer Institute of Canada (to A. M. E., and L. F.) and from the Medical Research Council of Canada (to A. M. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a studentship from the Medical Research Council of Canada.

These authors contributed equally to this work.

**
Research Scientist of the National Cancer Institute of Canada and supported by funds provided by the Canadian Cancer Society.

§§
Research Scholar of the Medical Research Council of Canada.

(^1)
The abbreviations used are: OBP, origin DNA-binding proteins; EBNA, Epstein-Barr virus nuclear antigen; MES, 4-morpholineethanesulfonic acid.

(^2)
H. Summers, J. A. Barwell, R. A. Pfuetzner, A. M. Edwards, and L. Frappier, submitted for publication.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.