From the Department of Biochemistry & Biophysics,
Texas A&M University, College Station, Texas 77843-2128 and
§ The Center for Structural Biology, Albert B. Alkek
Institute of Biosciences and Technology, Houston, Texas 77030-3303 and
The Center for Extracellular Matrix Biology, Texas A&M
University System Health Science Center, Albert B. Alkek Institute of
Biosciences and Technology, Houston, Texas 77030-3303
Received for publication, November 6, 2000, and in revised form, January 2, 2001
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ABSTRACT |
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The outer surface protein C (OspC) is one
of the major host-induced antigens of Borrelia burgdorferi,
the causative agent of Lyme disease. We have solved the crystal
structure of recombinant OspC to a resolution of 2.5 Å. OspC, a
largely Lyme disease is caused by a family of closely related spirochetes
referred to as Borrelia burgdorferi sensu lato. The organism can be transmitted from one mammal to another by Ixodes
ticks that require blood meals at three different stages of their life cycle (1). B. burgdorferi has a remarkable ability to adapt to life both in a mammal and an arthropod host. If not successfully treated, Lyme disease and the causative agent can persist for decades
in man, demonstrating that the spirochete can effectively combat or
avoid the host defense systems. At least one means of avoidance is by
modification of surface proteins. Surface proteins on
Borrelia are presumably directly involved in interactions
with molecules of the host. These surface proteins are mostly
lipoproteins and several mechanisms for regulating their expression
have been described (2). Analyses of the B. burgdorferi
genome suggest that the organism has the potential of producing more
than 100 different lipoproteins that could become anchored to the outer membrane (3). One of these surface proteins,
OspC,1 is immunodominant in
the early stages of Lyme disease (4). OspC is a basic protein
(isoelectric point ~9.0) with a subunit molecular weight of 20 kDa.
It is normally not produced while the spirochete is in the arthropod,
but can be detected on the Borrelia in the gut of feeding
ticks and in a mammalian host (5-9). The OspC expression is carefully
regulated possibly in a temperature-dependent manner (6).
The presence of OspC has been proposed to facilitate the migration of
B. burgdorferi from midgut to the salivary glands of ticks
and thereby participates in the transmission of the spirochete from
tick to the mammalian host (10). These attributes make OspC an obvious
candidate for vaccine development and initial studies indicate that
vaccination with recombinant OspC can, in fact, protect mice against
Lyme disease (11-13). However, the extent of the protection could be
limited by factors such as strain dependence (14) and the manner of
antigen preparation (10, 15-16). Mapping of protective epitopes on
OspC has suggested that these are conformation-dependent and may be located at several sites in the OspC protein (15). Alignment
of several known OspC sequences derived from different organisms of
B. burgdorferi sensu lato suggest that conserved regions are
interspersed with variable regions (17, 18). These conserved regions
have been targeted in immunodiagnostic approaches for Lyme disease
detection (19).
In this study, we report the crystal structure of OspC as a prelude to
structure-based development of peptide vaccines against Lyme disease.
Protein Expression and Purification--
Initial experiments
were conducted with a recombinant form of OspC covering the entire
coding region of the mature form of OspC from B. burgdorferi
strain N40 (20). The target sequence was obtained by the polymerase
chain reaction using Taq DNA polymerase and ligated into a
modified form of the pQE-30 expression vector (Qiagen Inc., Valencia,
CA). To enable cleavage of the N-terminal His6-tag, a
thrombin (trypsin) cleavage site was introduced immediately following
the histidine encoding region using the BamHI and
SacI restriction sites of the vector. DNA sequence analysis
confirmed that the resulting OspC nucleotide sequence was
identical to that of the published sequence. The purified protein
formed crystals after several months. However, analyses of the
crystallized protein by N-terminal sequencing and MALDI-MS revealed
that the first 13 N-terminal residues
(Cys19-Asn31) had been cleaved off.
A second expression construct was therefore made that lacked these
residues and started at residue Ser32. The resulting
construct (B.b. rOSPC) was transformed into JM101 cells
(Stratagene, La Jolla, CA), which were grown at 37 °C in Luria Broth
containing 100 µg/ml ampicillin. When the cell culture reached an
optical density of A600 = 0.6, isopropyl-
A third protein construct was engineered to allow MAD experiments using
a selenomethionine (SeMet) derivative. Because OspC does not contain
any methionines, residues Leu111 and Leu119
were mutated to Met. DNA sequencing of the resulting clone confirmed the correct incorporation of the methionine mutations and indicated an
unintentional mutation of Asn116 to Leu. After transforming
this plasmid into the methionine autotroph B843(DE3) cells (Novagen,
Madison, WI), selenomethionated OspC was produced by standard methods
(21) and purified using the same procedure as described for the native protein.
Crystallization--
Initial crystals were obtained using the
hanging drop method in condition no. 43 of the Crystal Screen I from
Hampton Research. High diffraction quality crystals were produced to a
size of ~0.6 × 0.4 × 0.1 mm in 10 mM Tris, pH
8.0, 5 mM dithiothreitol, 12% (w/v) polyethylene glycol
Mr 1,500 and with a protein concentration of
10-15 mg/ml. After soaking in cryoprotectant solution (10 mM Tris, pH 8.0, 5 mM dithiothreitol, 10%
glycerol, 15% polyethylene glycol Mr 400, 20%
polyethylene glycol Mr 1,500) or paratone oil for 1 min, the crystals were flash cooled to 100 K. Crystals of the
native protein were observed in the space group
P21212 with a = 31.9 Å, b = 46.9 Å, and c = 110.1 Å,
whereas the selenomethionine derivative formed crystals of the space
group P21 with a = 28 Å,
b = 45 Å, and c = 102 Å.
Structure Determination--
Native data were collected to 2.6 Å resolution on a MacScience image-plate-detector and using a RIGAKU
rotating anode generator equipped with osmic mirrors. Initial phases
were determined by MAD (22) using a SeMet derivative of OspC. MAD data
were collected at beamline 14-BM-D at the Advanced Photon Source (APS),
Argonne National Laboratory to 3 Å resolution on a charge-coupled
device area detector (ADSC Q4) at three wavelengths near the selenium absorption edge (see Table I). All native
and MAD data were processed and scaled using DENZO and SCALEPACK (23).
The program SOLVE (24) was used to locate the four selenium sites.
Phases obtained from SOLVE (mean figure of
merit 65 to 3.5 Å) were improved by solvent flattening and
noncrystallographic symmetry (NCS) averaging as implemented in DM
(Density Modification, Ref. 25) and CNS (Crystallographic & NMR System,
Ref. 26). Two molecules forming a dimer were found per asymmetric unit.
The molecular coordinates were constructed using NCS-averaged and
phase-combined electron density maps with the computer program O (27).
The structure was refined with CNS (26) to an R factor of
28% (Rfree = 34%). At this stage the model for
one subunit was used to obtain molecular replacement solution of the
native P21212 space group using CNS and data to
3.0 Å resolution. The final solution had an R-factor of
0.36 after rigid body refinement. Reflections up to 2.5 Å were included in subsequent refinement, and the structure was fit using 2|Fo|-|Fc| The OspC crystal structure was refined to 2.5 Å resolution and a
final R factor of 19.6% (Rfree of 26.2%). Both
the N- and C-terminal regions of the protein appear to be quite
flexible in OspC as we were unable to produce crystals unless the first 13 residues were removed from the initial recombinant protein. In
addition, the first eight N-terminal and the last six C-terminal residues from the truncated protein could not be located in the electron density maps. The electron density for the rest of the protein
was quite well resolved in the SeMet MAD electron density maps. The
final model showed excellent fit to omit electron density maps (Fig.
1) and exhibited good stereochemistry
with over 90% of the residues in the most favored regions and none in
the generously allowed or disallowed regions.
-helical protein, is a dimer with a characteristic central
four-helical bundle formed by association of the two longest helices
from each subunit. OspC is very different from OspA and similar to the
extracellular domain of the bacterial aspartate receptor and the
variant surface glycoprotein from Trypanosoma brucei. Most
of the surface-exposed residues of OspC are highly variable among
different OspC isolates. The membrane proximal halves of the two long
-helices are the only conserved regions that are solvent accessible.
As vaccination with recombinant OspC has been shown to elicit a
protective immune response in mice, these regions are candidates for
peptide-based vaccines.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-thiogalactoside was added to 1 mM to induce recombinant protein expression. Two hours
later, cells were harvested by centrifugation and lysed by sonication
and french press in a buffer containing 10 mM Tris, pH 8.0, 1 mM EDTA, and 0.1 M NaCl. The homogenate was
centrifuged for 15 min at 15,000 × g, and the
supernatant was applied at 4 °C to a Ni2+ charged
HiTrap-chelating column (Amersham Pharmacia Biotech). Protein was
eluted with an imidazole gradient, and fractions containing OspC were
pooled and dialyzed overnight against 10 mM Tris, pH 8.0, 0.1 M NaCl, 1 mM EDTA, and 5 mM
dithiothreitol. OspC (10 mg/ml) was incubated with trypsin (2 mg/ml) at
37 °C for 2 h to cleave off the His tag before the reaction was
stopped by the addition of 10 µl of 0.1 M
phenylmethylsulfonyl fluoride. Subsequently, pooled OspC fractions were
loaded onto a HiLoad Superdex-HR 75 column (Amersham Pharmacia Biotech)
equilibrated with 10 mM Tris, pH 8.0, 0.1 M
NaCl, 1 mM EDTA, and 5 mM dithiothreitol.
Fractions containing the purified protein were pooled and concentrated
to 30 mg protein/ml and used for the crystallization trials.
c and
composite omit electron density maps calculated with CNS. Backbone
geometry of the final model did not have any residue in the disallowed
regions as verified by PROCHECK (28). A summary of the relevant data
set statistics used in the structure determination and the
final refined statistics are shown in Table I.
Crystallographic and refinement statistics
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (45K):
[in a new window]
Fig. 1.
Example of the electron density. The
structure of helix 2 is shown with the composite omit density
(unweighted) contoured at 1.4
. The figure has been prepared using
the programs SwissPdbViewer (38) and
POV-Ray.2
In native P21212 crystals, OspC
crystallizes such that a biological dimer (70 Å × 50 Å × 30 Å) is
generated by a 2-fold crystallographic axis. A single subunit of OspC
is composed of five parallel -helices and two short
-strands
(Fig. 2, a and b).
Two long helices,
1 (residues 43-74) and
5 (residues 169-196),
from each subunit pack together to form a core four-helix bundle that
extends nearly the full-length of the protein. These
-helices pack
closely together such that N and C termini, which define the membrane
proximal end of both subunits, are in close proximity to each other. At the membrane distal end, however, the four helices diverge. The remaining helices,
2 (residues 94-112) and
3 (residues
119-144), together with an additional short helix
4 (residues
151-157) define the outer surface-exposed region. The residues
connecting the helices
1 and
2 form two short anti-parallel
-strands,
1 (residues 78-82) and
2 (residues 85-89), which
reside atop the surface-exposed end of OspC (Fig. 2b).
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The dimer interface of OspC buries ~22% of the accessible surface
area of each subunit. The buried surface is composed primarily of
hydrophobic residues. At the proximal end, residues contributing to the
subunit interactions (Ile49, Asn53,
Leu57, Lys60, Glu61,
Leu197) are located in the helices 1 and
5. At the
membrane distal end, most of the interactions involved in dimerization
are from the small
-sheet, which packs against the helices
2 and
3. Specifically, the sidechain of residue Ile81 is
embedded in a hydrophobic pocket formed by residues Leu96,
Leu137, Leu144, Leu149, and
Tyr101 of the other subunit. In addition, Leu86
interacts with Tyr101 and Ala102 of the other
subunit. The alignment of several OspC sequences (Fig.
3a) reveals that most residues
supporting the dimer formation are highly conserved. In addition,
several surface-exposed residues from the membrane proximal end of the
two long
-helices are conserved (Fig. 3b). Analytical
ultracentrifugation experiments performed at the Protein Interaction
Facility, University of Utah (Salt Lake City, UT; data not shown) and
preliminary NMR data (29) confirm that recombinant OspC is able to form
dimers in solution. Furthermore, Zückert et al. (30)
recently reported that the recombinant and the native lipoprotein are
dimeric.
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The surface of OspC contains several ionizable residues that impart a
distinct electrostatic potential at each face as shown in Fig.
3c. Ten glutamate and
aspartate residues make a highly electronegative surface on the face
formed by helices 3 and
4. This region is surrounded by several
lysine residues on either side. The alternating charged areas on the
surface of the protein are perhaps important for orienting the OspC
dimers so that they can form multimers or even lattices on the
spirochetes surface. The surface adjacent to the dimer interface shows
a highly negatively charged cavity (Fig. 3c), which might be
involved in protein-protein or protein-ligand interactions, although a
ligand for OspC has not been identified to date. Toward the membrane
proximal region, the surface is positively charged because of the
presence of several conserved lysine residues in helices
1 and
5
(Fig. 3). With the exception of a small pocket formed by residues
Lys92, Asp147, and Asn148, the
putative membrane distal region of OspC is less polar in nature. Most
of the residues lining this region of the OspC molecule are located in
the variable sequence regions and thus, may not be critical for a
conserved function.
The overall fold of OspC is very different from that of OspA, the only
other outer surface lipoprotein from Borrelia with known
structure. OspA is composed almost entirely of -strands (31). This
suggests that structural diversity of the outer surface antigens may
contribute to the ability of Borrelia to generate variability on its surface. Whereas no structural homologs of OspC
could previously be identified in the Protein Data Bank based on its
primary sequence alone, using the three-dimensional structure, we
identified two other surface proteins that are structurally similar to
OspC. OspC shows the most resemblance to the ligand-binding domain of
the Salmonella typhimurium aspartate receptor (32), a
receptor involved in signaling and chemotaxis (Fig. 2,
c and d). One of
the major structural differences between the two proteins is in the way
they associate with the membrane; OspC anchors to the membrane by its
covalently attached lipid, and the aspartate receptor has a
transmembrane domain (33). The extracellular domain of the aspartate
receptor shares only 15% sequence identity with OspC, but adopts a
very similar structure consisting of a homodimer where two four-helical
antiparallel bundles associate to form a quasi-four-helical bundle
(Fig. 2, c and d). The two structures show a root
mean square deviation of 2.4 Å upon superimposition of 86 C
atoms
of a single subunit. The residues forming the aspartate binding pocket
in the aspartate receptor are located at the distal end of the two long
helices and are contributed by both subunits. Whereas none of these
residues are conserved in OspC, the location of the aspartate binding
site is analogous to the charged cavity observed on the OspC surface.
The side chains of residues Lys60, Glu63,
Asp70, and Glu71 located on helix
1 of one
subunit, and residues Glu61 and Lys109 of the
second subunit line this charged cavity that forms near the dimer
interface. All of the residues are strictly conserved in the different
Borrelia strains. It is tempting to speculate that this
pocket may be responsible for recognition of a yet unknown ligand of
OspC. The aspartate receptor lacks the region containing two
-strands and, consequently, the two subunits exhibit a less extensive interface. The dimer interface of the aspartate receptor buries no more than 9% of the total accessible surface area of each
subunit. The weaker interaction has been proposed to facilitate the
intersubunit rotation/translation upon binding of aspartate necessary
for chemotactic signaling (32). In OspC, however, interactions of the
-strands with helices appear to lock the dimer into a much more
rigid conformation.
Another protein with a similar structure to OspC is the variant surface
glycoprotein (vsg) from Trypanosoma brucei (34). Interactions of the two subunits in this dimeric surface antigen is
also formed by two -helices and a small
-sheet. It is not clear
whether the presence of the small
-strands has an additional functionality beyond stabilization of the dimers. OspC has significant amino acid sequence homology to variable major proteins (vmp) from the
relapsing fever causing Borrelia hermsii and Borrelia turicatae (35). Conserved residues are primarily in the first two
-helices, including all of the amino acids that form the putative
binding pocket previously described. Furthermore, circular dichroism
spectra of recombinant forms of the two proteins are indistinguishable
(30). Thus, it would not be surprising if the vmps share similar
structures and functions. It is noteworthy that some members of the vmp
family have been previously shown to resemble the trypanosomal vsg
proteins (36) suggesting that the relapsing fever spirochetes adopt
strategies of antigenic variation similar to what have been observed
for trypanosomes. Trypanosomes move silent copies of the vsg
gene to the expression site, and in a similar way the spirochetes move
the vmp genes. Therefore, the structural similarity observed
between the OspC-vmp and vsg families, though minimal, points to
possible functional and mechanistic similarities in the two pathogens.
Recent reports have identified the protective epitope for OspC in different strains of B. burgdorferi sensu lato strains (15, 37). In the case of B. garinii, only the C-terminal end was shown to be responsible for immunological recognition (37) whereas for B. burgdorferi immunological studies demonstrated that the first 7 N-terminal or last 13 C-terminal residues were essential for antibody recognition and the binding was conformational-dependent (15). The adjacent location of N- and C-terminal residues in the membrane proximal region of OspC explains how the termini can be recognized by a single antibody. Because these residues are neither well ordered in the crystal structure nor show any significant propensity for a secondary structural element from predictions (data not presented), their interdependence for antibody recognition is probably because the overall fold brings the termini in close proximity to each other.
Based on the structure and the aligned Borrelia sequences,
most of the variable regions are found on the -strands and the two
loops connecting helices
2 with
3 and helices
3 with
4 (Fig. 3a). These regions are located at the putative
membrane distal end of the protein, far away from the identified
protective epitopes. Obviously these regions would be more accessible
to antibodies. However, conserved regions of the protein are also found
on the highly ordered and accessible surface of the OspC structure.
Helices
1 and
2 are of particular interest, based on their high
degree of conservation and this aforementioned resemblance to a ligand
binding cavity. Immunization experiments with synthetic peptides from
these regions could contribute to the development of a Lyme disease vaccine.
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ACKNOWLEDGEMENTS |
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We thank the APS beamline operators for assistance at beamline 14-BM-D, Julie C. Holding for excellent assistance in the laboratory, and Dr. Rebecca Rich for performing the analytical ultracentrifugation experiment.
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FOOTNOTES |
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* This work was supported by the Welch Foundation, the National Institutes of Health, and the Wenner-Gren Foundations, Sweden.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1G5Z) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). .
¶ Present address: Aventis Pharma Deutschland GmbH, Chemical Research-Molecular Modeling, Bldg. G838, D-65926 Frankfurt am Main, Germany.
** Present address: LifeCell Corp., One Millenium Way, Branchburg, NJ 08876.
To whom correspondence should be addressed. Tel.:
1-979-8627636; Fax: 1-979-8627638; E-mail: sacchett@tamu.edu.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M010062200
2 Contact author for further information about graphics software.
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ABBREVIATIONS |
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The abbreviations used are: OspC, outer surface protein C; vsg, variant surface glycoprotein; vmp, variable major proteins; MAD, multiwavelength anomalous dispersion.
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