Crystal Structure of Lyme Disease Antigen Outer Surface Protein C from Borrelia burgdorferi*

Christoph EickenDagger §, Vivek SharmaDagger §, Thomas KlabundeDagger §, Rick T. Owens||**, Dagmar S. Pikas||, Magnus Höök||, and James C. SacchettiniDagger §DaggerDagger

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha -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 alpha -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

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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-beta -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.

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|phic 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.

                              
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Table I
Crystallographic and refinement statistics


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.


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Fig. 1.   Example of the electron density. The structure of helix alpha 2 is shown with the composite omit density (unweighted) contoured at 1.4 sigma . 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 alpha -helices and two short beta -strands (Fig. 2, a and b). Two long helices, alpha 1 (residues 43-74) and alpha 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 alpha -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, alpha 2 (residues 94-112) and alpha 3 (residues 119-144), together with an additional short helix alpha 4 (residues 151-157) define the outer surface-exposed region. The residues connecting the helices alpha 1 and alpha 2 form two short anti-parallel beta -strands, beta 1 (residues 78-82) and beta 2 (residues 85-89), which reside atop the surface-exposed end of OspC (Fig. 2b).


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Fig. 2.   The overall structures of OspC and the aspartate receptor from Salmonella typhimurium. a, ribbon representation of the OspC dimer. The two long helices from each subunit form the core of the structure and extend the entire length of the molecule. b, a view of OspC along the 2-fold symmetry axis. The membrane distal end shows how the beta -strands contribute to the dimer interface. c, similar representation of the extracellular domain of the aspartate receptor from Salmonella typhimurium as OspC in a. Unlike OspC, the aspartate receptor core four-helix bundle does not diverege at the membrane distal end. d, in contrast to OspC, the membrane distal region of the aspartate receptor has fewer contacts at the dimer subunit interface. The alpha -helices are shown in blue, and the beta -strands are shown in yellow. The figures have been prepared using the programs SwissPdbViewer (38) and POV-Ray.2

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 alpha 1 and alpha 5. At the membrane distal end, most of the interactions involved in dimerization are from the small beta -sheet, which packs against the helices alpha 2 and alpha 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 alpha -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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Fig. 3.   Sequence alignment, conserved residues and electrostatic potential in OspC. a, sequence alignment of OspC from different Borrelia strains. Sequences were aligned by ClustalW (39). Strictly conserved residues are shaded, and residue positions are numbered according to the OspC_N40 strain. Sequence of recombinant OspC (rOSPC) used in this study is listed at the top. Triangles mark the two leucine positions that were mutated to methionine to produce the selenomethionine derivative. Secondary structure elements observed in the OspC structure are shown below the sequences. Bb, Borrelia burgdorferi; Bg, Borrelia garinii; Ba, Borrelia afzelii. This figure was generated by ALSCRIPT (40). b, accessible surface contours of the OspC dimer, where the positions of conserved residues are shown in blue. The figure was made with SPOCK (41). c, electrostatic potential on the molecular surface of the OspC dimer. The orientation on the left is identical to that in Fig. 2a, whereas the one on the right model is rotated 90 ° around the dimer 2-fold axis. The surface is colored according to the electrostatic potential with basic regions shown in blue, and acidic regions are in red. Solvent was excluded during the building of the molecular surface in the program SPOCK (41).

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 alpha 3 and alpha 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 alpha 1 and alpha 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 beta -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 Calpha 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 alpha 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 beta -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 beta -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 alpha -helices and a small beta -sheet. It is not clear whether the presence of the small beta -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 alpha -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 beta -strands and the two loops connecting helices alpha 2 with alpha 3 and helices alpha 3 with alpha 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 alpha 1 and alpha 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger Dagger 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.

    ABBREVIATIONS

The abbreviations used are: OspC, outer surface protein C; vsg, variant surface glycoprotein; vmp, variable major proteins; MAD, multiwavelength anomalous dispersion.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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