(Received for publication, May 12, 1995 )
From the
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
2
2
and had cell dimensions
a = 59 Å, b = 66.9 Å, and c = 69.8
Å with two molecules in the asymmetric unit.
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) ()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.
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.
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
-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
-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
-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. (
)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 CuK
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
, and using a protein density of
1.3 g/cm
, 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 = 72.80; C
= 100.00;
= 2.08;
= 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
2
2
. 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
2
2
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
P22
2
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
= 7.34; C
= 100.00;
= 3.67;
= 180.
Native data sets were
collected from the P22
2
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.