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Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi

2001; Springer Nature; Volume: 20; Issue: 5 Linguagem: Inglês

10.1093/emboj/20.5.971

1460-2075

D. Kumaran, S. Eswaramoorthy, Benjamin J. Luft, Shohei Koide, J J Dunn, Catherine L. Lawson, S. Swaminathan,

Heat shock proteins research

Article1 March 2001free access Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi D. Kumaran D. Kumaran Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author S. Eswaramoorthy S. Eswaramoorthy Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author B.J. Luft B.J. Luft Division of Infectious Diseases, School of Medicine, State University of New York, Stony Brook, NY, 11974 USA Search for more papers by this author S. Koide S. Koide Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, 14642 USA Search for more papers by this author J.J. Dunn J.J. Dunn Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author C.L. Lawson Corresponding Author C.L. Lawson Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Department of Chemistry, Rutgers University, 610 Taylor Road, Piscataway, NJ, 08854 USA Search for more papers by this author S. Swaminathan Corresponding Author S. Swaminathan Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author D. Kumaran D. Kumaran Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author S. Eswaramoorthy S. Eswaramoorthy Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author B.J. Luft B.J. Luft Division of Infectious Diseases, School of Medicine, State University of New York, Stony Brook, NY, 11974 USA Search for more papers by this author S. Koide S. Koide Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, 14642 USA Search for more papers by this author J.J. Dunn J.J. Dunn Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author C.L. Lawson Corresponding Author C.L. Lawson Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Department of Chemistry, Rutgers University, 610 Taylor Road, Piscataway, NJ, 08854 USA Search for more papers by this author S. Swaminathan Corresponding Author S. Swaminathan Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA Search for more papers by this author Author Information D. Kumaran1, S. Eswaramoorthy1, B.J. Luft2, S. Koide3, J.J. Dunn1, C.L. Lawson 1,4 and S. Swaminathan 1 1Biology Department, Brookhaven National Laboratory, Upton, NY, 11973 USA 2Division of Infectious Diseases, School of Medicine, State University of New York, Stony Brook, NY, 11974 USA 3Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, 14642 USA 4Department of Chemistry, Rutgers University, 610 Taylor Road, Piscataway, NJ, 08854 USA *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2001)20:971-978https://doi.org/10.1093/emboj/20.5.971 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Outer surface protein C (OspC) is a major antigen on the surface of the Lyme disease spirochete, Borrelia burgdorferi, when it is being transmitted to humans. Crystal structures of OspC have been determined for strains HB19 and B31 to 1.8 and 2.5 Å resolution, respectively. The three-dimensional structure is predominantly helical. This is in contrast to the structure of OspA, a major surface protein mainly present when spirochetes are residing in the midgut of unfed ticks, which is mostly β-sheet. The surface of OspC that would project away from the spirochete's membrane has a region of strong negative electrostatic potential which may be involved in binding to positively charged host ligands. This feature is present only on OspCs from strains known to cause invasive human disease. Introduction Lyme disease is the most common vector-borne disease in the United States and Europe (Barbour and Fish, 1993). It is a progressive multisystem disorder which begins at the site of a tick bite, producing a primary infection that can spread to secondary sites early in infection. Secondary sites may include the nervous system, heart and joints (van der Linde et al., 1990; Steere, 1991; Hansen and Lebech, 1992). The causative agent Borrelia burgdorferi induces a strong humoral response against endoflagellar protein, p41, and a protein constituent of the protoplasmic cylinder, p93, both of which are enveloped within the outer membrane, and some outer surface lipoproteins (Osps), which are major membrane components (Burgdorfer et al., 1982; Craft et al., 1986; Schwan et al., 1995; Schwan and Piesman, 2000). Experimental OspA and OspC vaccines have limited utility since they are usually only effective against challenge by the same strain and not by heterologous strains (Fikrig et al., 1990, 1992; Simon et al., 1991; Golde et al., 1995; Gilmore et al., 1996; Probert et al., 1997). Borrelia burgdorferi expresses OspA but not OspC when residing in the midgut of unfed ticks. However, when the tick starts feeding on mammals, OspC synthesis is induced and OspA is repressed (Schwan et al., 1995; Stevenson et al., 1995). The switch is in part due to the change in temperature; OspC is induced at 32–37°C, but not at 24°C, and this upregulation is at the transcriptional and translational levels (Tilly et al., 1997; Ramamoorthy and Philipp, 1998). Evidence suggests co-regulation of these two genes at the mRNA level (Jonsson and Bergstrom, 1995). Clearly, to survive in both hosts, spirochetes have evolved mechanisms for sensing the different host environments and responding accordingly. The ospC gene is located on a 27 kb circular plasmid and encodes a lipoprotein of 22–23 kDa (Marconi et al., 1993; Sadziene et al., 1993). The protein is initially synthesized with an 18-amino-acid-long signal sequence which is removed during processing and lipidation at the amino proximal Cys residue. OspC proteins are highly polymorphic and this variability extends even to strains collected from a single geographical area. For example, alleles of OspC collected from a single site on Shelter Island, NY, could be clustered into 19 major groups or types (A–S) based on DNA sequence homology (Wang et al., 1999). Sequence variation within a major group is <1% but ∼15% across the major groups. Variation within a local population is comparable to the variation of similar size samples collected from the entire species. This variability has consequences in the development of OspC-based seradiagnostic antigens and anti-OspC vaccines. Of the 19 major groups, only four (A, B, I and K) contain invasive clones and cause infections of skin and extracutaneous sites, while the others are non-human pathogens or infect only the skin (Seinost et al., 1999). However, the biological function of OspC is not known, and the relationship between OspC type and pathogenicity is not understood. In order to develop an effective OspC-based vaccine, it is important to know representative three-dimensional structures of at least a few OspCs, especially those from the invasive strains. This information could be useful for rational design of an OspC-based recombinant vaccine. Here we report the crystal structure of OspC from strain HB19, a member of invasive group I, and compare it with the crystal structure of OspC from strain B31 of group A. Results and discussion Structure determination The N- and C-truncated OspCs (residues 38–201) of strains HB19 and B31 of B.burgdorferi were cloned and expressed as described in Materials and methods. The, crystal structure of HB19 was solved using 2.8 Å resolution multiwavelength anomalous diffraction (MAD) data phasing with selenomethionine (SeMet)-labeled protein. Since there are four molecules per asymmetric unit, the MAD phases were further improved by non-crystallographic symmetry (NCS) averaging and density modification. The model building and initial refinements were completed with this improved set of phases. Further refinements were carried out with single wavelength 1.8 Å resolution data from a second crystal form of SeMet-labeled protein after orienting and positioning the molecule in the new unit cell by the molecular replacement method. In the final refinement cycle the NCS restraint was completely released. The model is complete except for the two N-terminal residues for which the electron density is very weak in all four monomers. The final model consists of 648 residues, 632 water molecules and six zinc ions. The crystallographic R-factor is 0.21 for 48 910 reflections in the resolution range 50–1.8 Å. The root mean square displacements (r.m.s.ds) between the NCS-related monomers are 99% purity could be obtained. SeMet-substituted OspC 38–201 HB19 was expressed in the E.coli 834(DE3) strain using minimal M9 media supplemented with L-SeMet and the other 19 L-amino acids (40 mg/l each). Selenomethionyl OspC 38–201 was purified using the same protocol as used for the native protein, except that egg white lysozyme (at 0.2 mg/ml) and EDTA (2.5 mM final concentration) were added to the resuspended cells to initiate lysis. Crystallization and data collection To determine the structure, SeMet-substituted protein was utilized. However, since there was only one internal methionine in a total of 164 residues, a mutant, Ile97Met, was generated by oligonucleotide-directed mutagenesis to increase the anomalous signal from selenium. Crystals of the SeMet OspC protein (aa 38–201) were grown with conditions similar to those for the native sample by vapor diffusion in sitting drops at 293K (Kumaran et al., 2001). Drops (6 μl) containing a 1:1 ratio of 6.9 mg/ml OspC and a precipitant containing 25% (v/v) PEG monomethyl ether 550, 10 mM zinc sulfate heptahydrate, 100 mM MES pH 6.5, were equilibrated against a reservoir containing 800 μl of the same precipitant. Long rectangular crystals with average dimensions of 0.3 × 0.3 × 1 mm3 grew in 1 week. Crystals were flash frozen after addition of 15% glycerol to the mother liquor. Diffraction data extending to 2.8 Å were collected from the frozen crystal with the B1 detector on beamline X12C at the National Synchrotron Light Source (NSLS). MAD data were collected around the selenium absorption edge (Ramakrishnan and Biou, 1997). The data were processed with DENZO and SCALEPACK (Otwinowski and Minor, 1997). Subsequently, single wavelength high-resolution data extending to 1.8 Å were collected with a better quality SeMet OspC crystal (Form II) on beamline X25 at the NSLS and were used in the later stages of refinement. Crystals of B31 OspC (aa 38–201) were grown at 4°C in 28–30% PEG 3350, 80 mM Tris–HCl pH 8.5, 100 mM MgCl2 and 18% glycerol, and diffraction was measured at NSLS beamline X12C (see Table II for diffraction data statistics). Table 2. OspC crystal, diffraction, phasing and model dataa HB19 (Form I) SeMet HB19 (Form II) SeMet B31 native Space group P21 P21 P1 Unit cell a (Å) 64.78 66.32 127.21 b (Å) 46.96 46.25 33.65 c (Å) 110.19 111.78 47.99 α (°) 84.04 β (°) 99.81 99.08 81.55 γ (°) 89.23 remote edge peak Wavelength (Å) 0.9300 0.9791 0.9794 0.9786 1.0920 Resolution range (Å) 50–2.8 50–2.8 50–2.8 50–1.8 50–2.5 Unique reflections 17 840 17 714 17 665 51 522 24 505 Completeness (%) 99.4 99.3 99.3 85.3 91.1 R-mergeb 0.083 0.079 0.117 0.087 0.043 12.2 14.6 Phasing powerc 1.2/1.4 1.5/1.5 FOMc, before density modification 0.44/0.33 PDB ID 1F1Me 1GGQ Content of asymmetric unit OspC monomers (residue range) 4 (40–201) 4 (40–201) solvent 632 112 ions 6 (Zn2+) 1 (Mg2+) Average B-value (Å2) main chain 22.1 43.2 side chain 27.0 45.2 solvent 31.5 30.5 Rworkd (reflections) 0.21 (48 910) 0.23 (22 087) Rfreed (reflections) 0.24 (2612) 0.27 (2418) R.m.s. bonds (Å) 0.004 0.005 R.m.s. angles (°) 1.010 1.130 φ–ψ, most favored regions (%)f 93 92 a HB19 and B31 were determined at Brookhaven National Laboratory and Rutgers University, respectively. b Rmerge = Σj(|Ih − h|)/ΣIh, where is the average intensity over symmetry equivalents. c Phasing power and FOM (figure of merit) are as defined in SHARP (De La Fortelle and Bricogne, 1997); centric/acentric reflection values are shown. d R = ΣFobs − Fcalc|/ΣFobs|; Rwork is summed over reflections used in refinement, Rfree is summed over reflections set aside for validation. e HB19 Form II data were used for refinement. f As defined in PROCHECK (Laskowski et al., 1993). Structure determination HB19. There are four molecules per asymmetric unit and each molecule has two SeMets. The selenium positions were obtained from Patterson and difference Fourier maps with the use of the PHASES program package (Furey and Swaminathan, 1997). As described elsewhere, there are two dimers in the asymmetric unit (Kumaran et al., 2001). A self-rotation function calculation revealed a strong peak of 56% of the origin peak corresponding to κ = 180° and a relatively weak peak of 25% of the origin peak at κ = 12°. Also, a native self-Patterson showed a peak (18% of the origin peak) at (0, 0, 1/2). Accordingly, the monomers are related by a non-crystallographic 2-fold axis forming a dimer, and the dimers are related by a pseudo translation of half the unit cell along the c axis related by a small rotation of 12°. A total of eight selenium atoms was input to the SHARP program to refine the phases (De La Fortelle and Bricogne, 1997). The resulting phases were further improved by NCS averaging in DM in two steps, averaging the non-crystallographic 2-fold related monomers first and then the dimers related by pseudo translational symmetry (Cowtan, 1994). The free R-factor after NCS averaging was 0.34. The resulting electron density map was of excellent quality and revealed an almost all-helical structure. Approximately 90% of the Cα chain was traced with the baton option in 'O' (Jones et al., 1991). The model building was completed using the two SeMets as markers. Further refinement was carried out with the high-resolution data set. Since the cell dimensions of the Form II crystal were slightly different from those of Form I, a rigid body refinement was initially carried out. During rigid body refinement, each of the four monomers was treated as a separate rigid group and gave an R-value and free-R value of 0.39 and 0.38, respectively. 5% of the data were set apart for the free-R calculation. B31. The B31 OspC crystal also contains two dimers in the asymmetric unit. The B31 structure was solved by the molecular replacement method with HB19 as a search model (Navaza and Saludjian, 1997). Both models were refined by the slow-cool annealing method (Brunger et al., 1998) alternating with model building until convergence. Composite omit maps clearly showed density for divalent cations and coordinating waters. The final model of HB19 contains four monomers of 162 residues each, six zinc atoms and 632 water molecules. Individual restrained B-factors were used in the final cycles of refinement for HB19 and B31. Two N-terminal residues were not included in the model for all the monomers because of poor electron density. The final refinement statistics are given in Table II. The HB19 and B31 OspC coordinates and experimental structure factors have been deposited with the Protein Data Bank [id codes 1F1M (HB19) and 1GGQ (B31)]. Acknowledgements We thank A.Glenn, B.Lade, H.Kycia and F.Mannino for technical assistance, and X.Yang for initial help with OspC purification and crystallization trials. We also thank X.Huang for his help. Research was supported by the National Institutes of Health (AI37256) at Brookhaven National Laboratory under contract with the US Department of Energy. B.J.L. was supported by the National Institutes of Health (#PO1-NS34092-01A2) and D.K. by the Veterans Administration Medical Center, Pittsburgh. References Barbour AG and Fish D (1993) The biological and social phenomenon of Lyme disease. Science, 260, 1610–1616.CrossrefCASPubMedWeb of Science®Google Scholar Brunger AT et al. (1998) Crystallography & NMR system: a new software suite for macr