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Journal of Bacteriology, February 2005, p. 1317-1323, Vol. 187, No. 4
0021-9193/05/$08.00+0     doi:10.1128/JB.187.4.1317-1323.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Putative Coiled-Coil Structural Elements of the BBA68 Protein of Lyme Disease Spirochetes Are Required for Formation of Its Factor H Binding Site

John V. McDowell,1 Matthew E. Harlin,1 Elizabeth A. Rogers,1 and Richard T. Marconi1,2*

Department of Microbiology and Immunology and Center for the Study of Biological Complexity,1 Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia2

Received 20 September 2004/ Accepted 23 November 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Factor H and factor H like-protein 1 (FHL-1) are complement regulatory proteins that serve as cofactors for the factor I-mediated cleavage of C3b. Some Lyme disease and relapsing fever spirochete species bind factor H to their surface to facilitate immune evasion. The Lyme disease spirochetes produce several factor H binding proteins (FHBPs) that form two distinct classes. Class I FHBPs (OspE orthologs and paralogs) bind only factor H, while class II FHBPs (BBA68) bind both factor H and FHL-1. BBA68 belongs to a large paralogous protein family, and of these paralogs, BBA69 is the member most closely related to BBA68. To determine if BBA69 can also bind factor H, recombinant protein was generated and tested for factor H binding. BBA69 did not exhibit factor H binding ability, suggesting that among family 54 paralogs, factor H binding is unique to BBA68. To identify the determinants of BBA68 that are involved in factor H binding, truncation and site-directed mutational analyses were performed. These analyses revealed that the factor H binding site is discontinuous and provide strong evidence that coiled-coil structural elements are involved in the formation of the binding site.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lyme disease is the most prevalent tick-borne zoonosis in North America, with ~24,000 cases reported to the Centers for Disease Control and Prevention in 2002. The etiological agents of Lyme disease are Borrelia burgdorferi, B. lonestari, B. garinii, and B. afzelii. Of these species only B. burgdorferi and B. lonestari are found in North America. All species have been detected in Europe and Asia except B. lonestari (7). It is evident from the chronic nature of Lyme disease that the Lyme disease spirochetes are able to evade immune destruction. Several potential mechanisms of immune evasion have been identified (22, 23, 33, 34, 36, 37). It has recently been demonstrated that some Borrelia species can bind the complement regulatory proteins factor H and or factor H like-protein 1 (FHL-1) to their surface (2, 18, 24, 29). Factor H and FHL-1 serve as cofactors for factor I, a serine protease that cleaves C3b to yield iC3b. C3b is a critical component of the complement cascade, and in the context of the alternative pathway, it plays an important role in opsonization. Factor H also functions to dissociate Bb from the C3 convertase complex, leading to the down-regulation of C3b production (35). The binding of factor H and FHL-1 by pathogens enhances the cleavage of C3b directly on the cell surface, thereby decreasing the efficiency of opsonization and phagocytosis. B. burgdorferi sensu lato strains produce two distinct classes of factor H binding proteins (FHBPs) (26). The class I FHBPs, which include the OspE paralogs and orthologs, bind only factor H. The class II proteins are of higher molecular weight, are unrelated to OspE, and bind both factor H and FHL-1. BBA68 is the only class II protein that has been identified to the sequence level (16) (also referred to as BbCRASP-1). The gene encoding BBA68 resides on a 54-kDa linear plasmid (lp54) and is part of a 14-member paralogous gene family designated by The Institute for Genomic Research as family 54. Paralogs of this gene family exhibit significant intrafamily divergence, with amino acid similarity values as low as 7.3% and identity as low as 5.4%. It is not yet known if other members of this family are able to bind factor H.

The interaction between factor H and Borrelia FHBPs has been the focus of several recent studies (1, 13, 14, 17, 24, 25, 26). Factor H and FHL-1 are glycoproteins comprised of a series of short consensus repeats (SCRs). The interaction of factor H with B. burgdorferi class I FHBPs is mediated specifically by SCRs 19 and 20 (38), while the binding of factor H and FHL-1 to the class II proteins is mediated by SCRs 6 and 7. However, the BBA68 determinants required for factor H and FHL-1 binding are less clear. Kraiczy et al. (16) demonstrated that the C-terminal region of BBA68 is required. However, the potential involvement of other domains of the protein in factor H binding was not assessed. Studies of factor H binding by OspE revealed that both the C and N termini are required for factor H binding (29). The goal of this study was to investigate the potential involvement of other domains of BBA68 in factor H binding by using multiple mutational strategies. These analyses demonstrate that the binding site for factor H is discontinuous and that its presentation is dependent on several predicted coiled-coil motifs that are distributed throughout BBA68. In addition, we also demonstrate that BBA69, the family 54 member that is most closely related to BBA68, does not bind factor H, suggesting that within family 54, factor H binding is unique to BBA68.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. All vectors and reagents for ligase-independent cloning (LIC) were obtained from Novagen. PCR reagents were from Roche. All DNA primers were from IDT, and DNA sequencing was performed by MWG Biotech. Factor H and anti-human factor H antisera were from Calbiochem. All secondary antibodies and chemiluminescent reagents were from Pierce. Polyvinylidene difluoride membranes were from Millipore. Precast Criterion 12.5% polyacrylamide gels were from Bio-Rad.

LIC and expression of recombinant proteins. This analysis focuses on BBA68, a class II FHBP of B. burgdorferi B31MI, and its most closely related paralog, BBA69. Note that in a recent analysis, BBA68 was also referred to as BbCRASP-1 (16). Based on the determined genome sequence for B31MI (10), primers (Table 1) were designed to amplify BBA69, BBA68, and subfragments of BBA68. The primers were designed with tail sequences to allow for LIC of the amplicons into the pET-32 Ek/LIC expression vector and subsequent expression as S-tag and His-tag fusion proteins (23). All procedures for PCR, cloning, and expression of recombinant proteins were as previously described (29). Note that all of the recombinant proteins carry an N-terminal S tag that adds ~17 kDa to the mass of each recombinant protein. All constructs, including that which we refer to as the full-length form, were generated without the leader peptide (~24 amino acids).


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  		TABLE 1. Primers employed in this study

 
Site-directed mutagenesis of putative BBA68 coiled-coil domains. Site-directed mutagenesis was conducted by using a two-step PCR-based approach with mutagenic primers listed in Table 1. All amplification reactions were performed using standard PCR conditions and Pfu polymerase. The template for the PCR reactions was a recombinant plasmid carrying BBA68, generated as described in this report. The gene was amplified as two separate products. For all constructs the 5' portion of the gene was PCR amplified using the A68-25(+) primer and a reverse mutagenic primer that harbored the desired sequence changes. The forward primer used to amplify the 3' half of the gene also contained the desired sequence changes and complemented the reverse primer used to amplify the 5' portion of the gene. The amplicons derived from each half of the gene were purified from an agarose gel and combined to serve as a template in another PCR reaction. Eight cycles of PCR were run in the absence of oligonucleotide primers. During these first eight cycles, the complementary sequences on the two template amplicons allow annealing to each other. Hence, each amplicon essentially serves as a large primer. After eight cycles, the BBA68-25(+) and BBA68-251(–) primers (which contain tails that allow cloning using the LIC approach with the pET32 Ek/LIC vector) were added to amplify the full-length mutated gene. The resulting amplicons were purified, the single-strand tails were generated by treatment with T4 DNA polymerase as directed by the vector manufacturer, and the amplicons were annealed with the linearized pET32 Ek/LIC vector. To propagate the plasmids, the annealed products were introduced into Escherichia coli NovaBlue DE3 cells by transformation and plated on Luria-Bertani plates containing 50 µg of ampicillin ml–1. To screen for recombinants, E. coli colonies were picked from the plate and boiled in 50 µl of H2O. The presence and size of the inserts in the recombinant plasmids were determined by PCR amplification. Protein expression was induced by growth of the cultures overnight at 37 °C (220 rpm). Induction with isopropylthiogalactoside was found to be unnecessary. After cultivation the cells were harvested by centrifugation and washed with phosphate-buffered saline. The mutant BBA68 proteins were then tested as described elsewhere in this report for expression of the fusion proteins and for their ability to bind factor H.

SDS-PAGE, immunoblot analyses, and factor H affinity ligand-binding immunoblot (ALBI) assays. The procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were as previously described (32). To verify expression of all recombinant proteins, cell lysates of induced cultures were resolved by SDS-PAGE and immunoblotted, and the membranes were screened with S-protein horseradish peroxidase (HRP) conjugate (1:20,000 dilution). The S-protein HRP conjugate binds to the N-terminal S tag present on all of the recombinant proteins. Binding of S-protein was detected by chemiluminescence using the Super-Signal West Pico kit. To determine if the various recombinant proteins investigated here bind factor H, factor H binding was employed as previously described (29). The concentration of factor H in the assay was 10 ng ml–1, and the anti-factor H antiserum was used at a dilution of 1:800. Rabbit anti-goat immunoglobulin G served as the secondary antibody (Ab) and was used at a dilution 1:40,000. Detection was by chemiluminescence as described above.

DNA sequence analyses and computer-assisted analysis of BBA68 and BBA69 structures. The sequences of all constructs and mutants analyzed in this report were determined by DNA sequence analysis. The integrity of the open reading frames was confirmed by translation of the determined sequences by using the TRANSLATE program. The predicted secondary structures of the BBA69, BBA68, and BBA68 mutants were determined by using the GOR program. The predicted probability of coiled-coil formation in all recombinants was assessed by using the COILS program (20). The COILS analysis was run without and with weighting (2.5) of the a and d positions of the coiled-coil heptad repeat using the MDIK matrix and windows of 21 and 28 amino acid residues.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of the ability of the family 54 paralogs, BBA69 and BBA68, to bind factor H. It had previously been demonstrated that BBA68 is able to bind factor H (16). BBA68 is one of 14 members of paralogous family 54 (10). Of these proteins, BBA69 is the most closely related to BBA68 with 58.3% sequence identity and 75.3% sequence similarity, and a pileup of the two sequences is shown in Fig. 1. The next most related paralog, BBI36, exhibits only 38.6% identity and 57.6% similarity. Based on this, we focused on BBA69 and sought to determine if it can bind factor H. Recombinant BBA68 and BBA69 were generated and tested using the factor H-ALBI assay (Fig. 2). While BBA68 (the positive control) readily bound factor H, BBA69 did not. This suggests that BBA68 is the only member of family 54 that can bind factor H.



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  		FIG. 1. Sequence alignment and secondary structure predictions for BBA68 and BBA69. The amino acid sequences of the family 54 paralogs, BBA68 and BBA69, are aligned. The secondary structure predictions are shown either above or below the corresponding sequence. The putative factor H binding regions identified by Kraiczy et al. are labeled as regions 1, 2, or 3, and the amino acid residues within each region are indicated by underlining. The predicted coiled-coil domains of BBA68 are indicated by boldfacing the corresponding residues in the secondary structure predictions and are labeled as CC1, CC2, or CC3.

 


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  		FIG. 2. Analysis of the ability of the family 54 paralog, BBA69, to bind factor H. Recombinant BBA68 and BBA69 were generated as S-tag fusion proteins as described in the text. Immunoblots of cell lysates of E. coli that had been induced with isopropyl-ß-D-thiogalactopyranoside were screened with S-protein HRP conjugate or tested for factor H binding by using the factor H ALBI assay. BBA68 served as the positive control.

 
Analysis of the ability of truncated BBA68 recombinant proteins to bind factor H. To localize the factor H binding site within BBA68, recombinant full-length and N- and C-terminally truncated proteins were generated in E. coli and tested for factor H binding ability (Fig. 3). The recombinant proteins were separated by SDS-PAGE, and several identical immunoblots were generated. Relatively equal expression of each protein in E. coli was demonstrated by screening one blot with S-protein HRP conjugate. To identify the minimum subfragment that can bind factor H, an identical blot was screened using the factor H ALBI assay. Full-length BBA68 and two N-terminal truncations with 49 or 73 amino acids deleted retained factor H binding ability. In contrast, deletion of the C-terminal 24 amino acids or N-terminal truncation of the first 98 amino acids resulted in the complete loss of factor H binding ability. The requirement for determinants located within the N- and C-terminal portions of the protein indicates that the factor H binding site is discontinuous and involves widely separated residues.



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  		FIG. 3. BBA68 constructs employed in this study and analysis of their ability to bind factor H. The schematic depicts the various BBA68 truncations that were generated. All constructs were generated as N-terminal S-tag, His-tag fusion proteins and lack the leader peptide. The nomenclature assigned to each truncation is indicated. To confirm expression of each recombinant protein in E. coli, an immunoblot of all constructs was screened with S-protein HRP conjugate. The ability of each construct to bind factor H, as indicated a + or – to the right of each construct, was assessed, using the factor H ALBI assay as described in the text.

 
Computer-based structural analysis of BBA68 and BBA68 mutants and construction of site-directed mutants. As an initial step in identifying structural elements that are required for the presentation of the discontinuous factor H binding site, computer-assisted structural analyses of BBA68 and BBA69 were performed (Fig. 1). Both proteins were found to have a high alpha-helical content. To determine if the alpha helices have the potential to form coiled coils, the COILS program was employed (20). Three predicted coiled-coil domains, designated coiled-coil 1 (cc1), coiled-coil 2 (cc2), and coiled-coil 3 (cc3), were identified. For ease of discussion cc3 is further subdivided into cc3a and cc3b (for the N- and C-terminal halves of cc3, respectively). To determine if the putative coiled coils of BBA68 are involved in factor H binding or in the formation of the factor H binding site, amino acid substitutions that were designed to either disrupt or have no effect on coiled-coil formation were introduced into BBA68. The predicted effect of each substitution is indicated in Table 2. To decrease the probability of coiled-coil formation, destabilizing residues (Ser, Trp, Lys, and His) were introduced into the a and d positions of the coiled-coil heptad repeat motif (19). Additional amino acid substitutions that were either conservative or at other positions of the heptad repeat and that were not predicted to disrupt coiled-coil formation were also introduced. The expression of each of the mutated recombinant proteins was confirmed by immunoblotting, using the S-protein HRP conjugate, and the ability of each to bind factor H was determined using the factor H ALBI assay (Fig. 4). Two cc1 mutants were generated, cc1m1 and cc1m2. In cc1m1, two Ile residues located at a and d positions were replaced with the destabilizing residues Lys and Ser. When using a 21-amino-acid window, the probability of coiled-coil formation dropped from 0.9 to 0.5. This mutated protein completely lost its ability to bind factor H. In contrast, when the same Ile residues were replaced with Val, the probability of cc1 formation was only slightly reduced, dropping to 0.75. This mutant retained full factor H binding ability. This set of experiments, as with the additional mutations discussed below, indicates that a strict primary sequence within cc1 is not required for factor H binding but rather that the formation of the factor H binding site is dependent on the formation of specific structural elements in BBA68.


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  		TABLE 2. Coiled-coil mutational analyses and coiled-coil predictions

 


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  		FIG. 4. Analysis of the ability of specific coiled-coil mutants to bind factor H. Lysates of E. coli expressing each recombinant protein were fractionated by SDS-PAGE and immunoblotted. The immunoblots were screened with S-protein HRP conjugate or tested for factor H binding by using the factor H-ALBI assay. All methods were as described in the text. Molecular mass markers are indicated.

 
More-extensive mutational analyses were performed for cc2 and cc3. In the cc2 mutant, cc2m1, centrally located Leu residues at the d and a positions and a Lys at an e position were substituted with the destabilizing residues, Ser, His, and Arg, respectively. The probability of formation of cc2 dropped from 0.996 to 0.044, and this mutant completely lost factor H binding ability. Similarly, replacement of the next consecutive Leu pair in cc2 with His residues reduced cc2 formation probability to 0.242. This mutant also lost factor H binding ability. In cc2m3, a Lys and Ile at the e and g positions were replaced with Asp and Thr, respectively. These substitutions did not decrease cc2 formation probability, and consistent with this, no loss in factor H binding was observed. The amino acid substitutions introduced into cc2m4 and cc2m5 were not predicted to significantly inhibit coiled-coil formation. These mutants retained factor H binding ability, albeit at a reduced level. Mutational analyses of cc3 were conducted on both the N- and C-terminal regions of the alpha helix that is predicted to form this coiled coil. We refer to the N-terminal part of cc3 as cc3a and the C-terminal part as cc3b. In the cc3am1 mutant, the Leu and Ile located at the a and d positions were replaced with Trp and Lys, respectively, resulting in the complete loss of factor H binding. The probability of cc3a formation in this mutant was reduced from 0.979 to 0.129 (using the 28-amino-acid-residue window). Note that the reason that the probability drop was minor when a 21-amino-acid window was used is because with this window size, the cc3am1 mutations lie outside of the 21-residue window. Mutants in the C-terminal portion of cc3 had less of an impact on factor H binding. Collectively, the site-directed mutational analyses suggest that widely separated coiled-coil structural elements of BBA68 are required for factor H binding and clearly indicate that the binding domain for factor H is not contiguous.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An extraordinarily diverse group of organisms, including viruses, bacteria, parasites, and tumor cells, exploit factor H binding to promote immune evasion, adherence, or intracellular localization (3, 4, 6, 8, 9, 11, 13, 14, 17, 24, 26-29, 31). In addition to their contribution to pathogenesis, the FHBPs are also of interest in vaccine development. This study was performed to identify the residues or structural elements of the B. burgdorferi class II FHBP, BBA68, that are required for the binding of factor H and to determine if BBA69 (another family 54 member) can also bind factor H.

BBA68 is one of 14 members of paralogous protein family 54 (10). This family is characterized by significant intrafamily divergence. The paralog most closely related to BBA68 is BBA69 (75.3% similarity and 58.3% identity). BBA69 encodes a 263-amino-acid protein with a predicted signal peptidase cleavage site at residue 25. The next most closely related paralog to BBA68, BBI38, exhibits only 38.4% identity with BBA68. Since BBA69 is the paralog most closely related to BBA68 and has features suggesting surface exposure, we analyzed the ability of this paralog to bind human factor H. With the factor H ALBI assay, no binding was detected. The inability of BBA69 to bind factor H is perhaps not surprising in view of the significant sequence divergence between these proteins. It is important to note that while the putative factor H interaction domain, region 3, identified by Kraiczy et al. (16), is conserved in BBA69, it does not convey factor H binding ability to BBA69. The potential contribution of these domains in factor H binding is discussed in detail below.

The molecular basis of the interaction between factor H and BBA68 was recently investigated (16), and it was demonstrated that BBA68 C-terminal truncations of 7 or 11 amino acids resulted in attenuated factor H binding or no factor H binding, respectively (16). From this it can be concluded that the C terminus of BBA68 is either directly or indirectly involved in factor H binding. Earlier truncation analyses of the OspE protein of the Lyme disease spirochetes also demonstrated that the C terminus is required for factor H binding. However, N-terminal truncations had the same effect, indicating that both the N and C termini of OspE are required for factor H binding (29). Prior to this report, the involvement of other regions of BBA68 in factor H binding had not been assessed. As a first step in addressing this, independent N- and C-terminal truncations of BBA68 were generated. Consistent with that reported by Kraiczy et al., truncation of the C terminus completely abolished factor H binding (16). While N-terminal truncations of 49 or 73 residues had no impact on factor H binding, removal of the first 98 residues resulted in the complete loss of binding. Hence, the minimum BBA68 fragment generated as part of this report that retained factor H binding ability spanned residues 73 through 251. The data presented here indicate that as with OspE, widely separated domains of BBA68 are required for or involved in factor H binding and that the binding is not mediated solely by C-terminal structural or linear determinants.

In spite of the shared ability to bind the same ligand (factor H), comparative sequence analysis of the FHBPs of the Borrelia revealed weak or no homology among these proteins. This observation suggests that factor H binding is not mediated by a conserved primary sequence element. However, computer-assisted structural analyses indicate that the Borrelia FHBPs are predicted to have coiled-coil structural motifs. Analysis of the predicted structure of FHBPs from other organisms, including the PspC protein of the pneumococcus (9) and Omp100 of Actinobacillus actinomycetemcomitans (6), revealed that they also have the potential to form coiled coils. Coiled-coil domains have been demonstrated to facilitate intra- and intermolecular protein-protein interactions. In general, these structural elements are defined by the presence of a heptad repeat of sequence (a-g)n where the a and d positions are usually hydrophobic residues (Leu, Ile, or Val). Residues at the e and g positions are usually charged or polar. The side chains of the a and d residues pack into a hydrophobic core, leaving the e and g residues exposed where they are available for ionic interactions or salt bridges. The interaction of factor H with some FHBPs is known at least in part to be a charge-mediated interaction, since it can be inhibited by heparin (17). Further evidence for a charge-based interaction comes from Ala scanning mutagenesis analyses that revealed that several Lys residues of OspE are required for factor H binding (1). Based on this, we hypothesized that the coiled-coil domains of BBA68 are involved in long-range intermolecular interactions that result in the proper presentation of a charged pocket that is required for factor H binding. To test this hypothesis, site-directed mutagenesis of each of the three putative coiled coils (cc1, cc2, and cc3a and cc3b) was performed. Each mutant had two or three amino acid substitutions introduced. Replacement of the a- and d-position residues of each coiled coil with destabilizing residues resulted in a significant decrease in the predicted probability of coiled-coil formation, and coincident with this, these mutants exhibited a complete loss of factor H binding ability. In contrast, the introduction of stabilizing residues at the a and d positions had no impact on factor H binding. It is striking that substitutions in widely separated domains of BBA68 lead to the complete loss of factor H binding, an observation that indicates that multiple domains of BBA68 are directly or indirectly involved in factor H binding.

The putative coiled coils of BBA68 are relatively short. In short coiled coils (<40 residues), the hydrophobic core is most stable when it is enriched with bulky nonpolar amino acids (Leu and Thr) and is less stable when the a and d positions are nonpolar residues with small side chains (19). For example, while Val is a stabilizing residue in extended coiled coils, in the context of short coiled coils, this residue typically decreases the predicted probability of coiled-coil formation. To determine if replacement of a- and d-position Ile residues with Val would lead to decreased probability of coiled-coil formation (a reflection of decreased stability) and thus attenuate factor H binding, Val residues were introduced into cc1 and cc2. The probability of cc1 formation in cc1m2 decreased by 18%, but these substitutions had no effect on factor H binding. In contrast, in cc2 the Val substitutions, which decreased the probability of cc2 formation in cc2m4 by 46%, resulted in at least a 10-fold reduction in factor H binding. This observation is consistent with the prediction that partial destabilization of the coiled coil would attenuate but not necessarily eliminate factor H binding. As controls for these analyses, several other mutations that were not predicted to impact on coiled-coil formation were also constructed and tested for factor H binding. As an example, the probability of cc2 formation in the cc2m3 mutant was unchanged as a result of the substitutions introduced, and consistent with this, no decrease in factor H binding was observed.

Earlier analyses of the BBA68 sequence revealed that the protein has three regions (referred to as regions 1, 2, and 3) (indicated in Fig. 1) that exhibit weak homology to the C-terminal domain of OspE (16). As discussed above, the C terminus of OspE is one of several domains of the protein that is involved in or required for factor H binding. In an earlier analysis, weak factor H binding to peptides spanning regions 2 and 3, but not region 1, was demonstrated (16). Regions 1 and 2 reside within cc2 and cc3, respectively. While region 1 peptides do not bind factor H, the mutational analyses presented above clearly demonstrate the requirement for the cc1 structural element in factor H binding. Hence, cc1 does not appear to directly interact with factor H but may be indirectly involved by participating in the formation or presentation of the discontinuous factor H binding site. In contrast to region 1, peptides corresponding to regions 2 and 3 have been shown to interact with factor H (16). However, as demonstrated by the mutational studies presented here, a strict region 2 primary sequence is not required, but rather it is the ability of this region to form a cc that is most important in factor H binding. Region 3 lies outside of the cc domains of BBA68 and maps within a C-terminal 12-residue alpha helix. Interestingly, the seven-amino-acid C-terminal deletion generated by Kraiczy that led to significantly attenuated factor H binding is not part of region 3, and peptides spanning the terminal 13 residues of BBA68 did not bind factor H. This is similar to that previously reported for the factor H-binding OspE protein. Metts et al. demonstrated that while the C terminus is required for factor H binding, peptides corresponding to the C terminus do not bind factor H (29). It is important to note that BBA69 carries a conserved copy of region 3 in its C terminus but does not bind factor H. Hence, while the region 3 sequence, in the context of full-length BBA68, may be required for factor H binding, it is not in and of itself sufficient to convey factor H binding. It appears that the C terminus of BBA68 (and most likely OspE) must be presented in the context of a specific conformation in order to allow for optimal factor H binding.

Coiled-coil structures are widespread in proteins with an estimated 3% of all alpha helices existing as this structural element (19). These motifs have been extensively studied as models for understanding protein-protein interactions. There is also a considerable body of literature demonstrating their involvement in viral membrane fusion events (15, 21). However, in spite of their widespread occurrence and the general assumption of their importance in protein-protein interactions, to our knowledge there have been only a few published studies that provide evidence that coiled coils are required for ligand binding in bacterial systems. We have recently demonstrated, using an approach similar to that presented here, that coiled-coil domains in the OspE protein of the Lyme disease spirochetes are required for the binding of factor H (25). Arthur et al. (5) introduced amino acid substitutions into coiled coils in the ß' subunit of E. coli RNA polymerase and found that these mutations eliminated its ability to bind sigma 70. Hence, the study presented here is among one of the first to directly demonstrate the importance of coiled coils in ligand binding by a bacterial protein. The information presented here can be exploited in the design of FHBP-based vaccinogens. One of the confounding problems with FHBP-based vaccinogens is that these proteins are likely to be rapidly saturated with highly abundant factor H upon injection and as such elicit a restricted Ab response due to masking by the large factor H molecule. The OspE protein of the Lyme disease spirochetes serves as a case in point. While this protein is antigenic and immunogenic (12, 29, 33), it does not elicit a protective Ab response (30), and the Ab response to the protein is to restricted domains (29). It is our hypothesis that modification of these proteins so that they no longer bind factor H will lead to the development of a less restricted, and potentially protective, Ab response. In addition, by blocking the ability of recombinant vaccinogen to bind factor H, antibody may also be elicited to the factor H binding sites of the protein. These antibodies may be able to block factor H binding by the native, pathogen-associated FHBP and thereby render the organism more susceptible to complement. Future analyses will seek to identify the specific residues of BBA68 that come in direct contact with factor H and will also seek to more broadly investigate the role of coiled coils in the interaction of other FHBPs with factor H.


   ACKNOWLEDGMENTS
 
This work was supported in part by grants from the NIAID, NIH (RO1AI37787 and R21 AI059257). J. V. McDowell was supported in part by a Molecular Pathogenesis training grant from NIAID.

We thank members of the Marconi lab and the Virginia Commonwealth University Molecular Pathogenesis Group for helpful discussions. J.V.M. also thanks Dominic Capodanno for insightful conversation about the study.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, P.O. Box 980678, Richmond, VA 23298-0678. Phone: (804) 828-3779. Fax: (804) 828-9946. E-mail: rmarconi{at}hsc.vcu.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Alitalo, A., T. Meri, T. Chen, H. Lankinen, Z.-Z. Cheng, T. Sakari Jokiranta, I. J. T. Seppala, P. Lahdenne, P. S. Hefty, D. R. Akins, and S. Meri. 2004. Lysine-dependent multipoint binding of the Borrelia burgdorferi virulence factor pouter surface protein E to the C terminus of factor H. J. Immunol. 172:6195-6201.[Abstract/Free Full Text]
  2. Alitalo, A., T. Meri, L. Rämö, T. S. Jokiranta, T. Heikkilä, I. J. T. Seppälä, J. Oksi, M. Viljanen, and S. Meri. 2001. Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation. Infect. Immun. 69:3685-3691.[Abstract/Free Full Text]
  3. Alitalo, A., T. Meri, H. Lankinen, I. Seppala, P. Lahdenne, P. S. Hefty, D. Akins, and S. Meri. 2002. Complement inhibitor factor H binding to Lyme disease spirochetes is mediated by inducible expression of multiple plasmid-encoded outer surface protein E paralogs. J. Immunol. 169:3847-3853.[Abstract/Free Full Text]
  4. Areschoug, T., M. Stalhammar-Carlemalm, I. Karlsosson, and G. Lindahl. 2002. Streptococcal beta protein has separate binding sites for human factor H and IgA-Fc. J. Biol. Chem. 277:12642-12648.[Abstract/Free Full Text]
  5. Arthur, T. M., L. C. Anthony, and R. B. Burgess. 2000. Mutational analysis of the B'260-309, a sigma 70 binding site located on Escherichia coli core RNA polymerase. J. Biol. Chem. 275:23113-23119.[Abstract/Free Full Text]
  6. Asakawa, R., H. Komatsuzawa, T. Kawai, S. Yamada, R. B. Goncalves, S. Izumi, T. Fujiwara, Y. Nakano, H. Shiba, M. A. Taubman, H. Kurihara, and M. Sugai. 2003. Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans. Mol. Microbiol. 50:1125-1139.[CrossRef][Medline]
  7. Baranton, G., D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont. 1992. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int. J. Syst. Bacteriol. 42:378-383.[CrossRef][Medline]
  8. Dave, S., S. Carmicle, S. Hammerschmidt, M. K. Pangburn, and L. S. McDaniel. 2004. Dual roles of PspC, a surface protein of Streptococcus pneumoniae, in binding human secretory IgA and factor H. J. Immunol. 173:471-477.[Abstract/Free Full Text]
  9. Duthy, T. G., R. J. Ormsby, E. Giannakis, A. D. Ogunniyi, U. H. Stroeher, J. C. Paton, and D. L. Gordon. 2002. The human complement regulator factor H binds pneumococcal surface protein PspC via short consensus repeats 13 to 15. Infect. Immun. 70:5604-5611.[Abstract/Free Full Text]
  10. Fraser, C., S. Casjens, W. M. Huang, G. G. Sutton, R. Clayton, R. Lathigra, O. White, K. A. Ketchum, R. Dodson, E. K. Hickey, M. Gwinn, B. Dougherty, J. F. Tomb, R. D. Fleischman, D. Richardson, J. Peterson, A. R. Kerlavage, J. Quackenbush, S. Salzberg, M. Hanson, R. Vugt, N. Palmer, M. D. Adams, J. Gocayne, J. Weidman, T. Utterback, L. Watthey, L. McDonald, P. Artiach, C. Bowman, S. Garland, C. Fujii, M. D. Cotton, K. Horst, K. Roberts, B. Hatch, H. O. Smith, and J. C. Venter. 1997. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390:580-586.[CrossRef][Medline]
  11. Giannikis, E., T. S. Jokiranta, D. A. Male, S. Ranganathan, R. J. Ormsby, V. A. Fischetti, C. Mold, and D. L. Gordon. 2003. A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein. Eur. J. Immunol. 33:962-969.[CrossRef][Medline]
  12. Hefty, P. S., C. S. Brooks, A. M. Jett, G. L. White, S. K. Wikel, R. C. Kennedy, and D. R. Akins. 2002. OspE-related, OspF-related and ELp lipoproteins are immunogenic in baboons experimentally infected with Borrelia burgdorferi and in human Lyme disease patients. J. Clin. Microbiol. 40:4256-4265.[Abstract/Free Full Text]
  13. Hellwage, J., T. Meri, T. Heikkila, A. Alitalo, J. Panelius, P. Lahdenne, I. J. T. Seppala, and S. Meri. 2001. The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi. J. Biol. Chem. 276:8427-8435.[Abstract/Free Full Text]
  14. Hovis, K., J. V. McDowell, L. Griffin, and R. T. Marconi. 2004. Identification and characterization of a linear plasmid encoded factor H-binding protein (FhbA) of the relapsing fever spirochete, Borrelia hermsii. J. Bacteriol. 186:2612-2618.[Abstract/Free Full Text]
  15. Joshi, S. B., R. E. Dutch, and R. A. Lamb. 1998. A core trimer of the Paramyxovirus fusion protein: parallels to influenza virus hemagglutinin and HIV-1 gp41. Virology 248:20-34.[CrossRef][Medline]
  16. Kraiczy, P., J. Hellwage, C. Skerka, H. Becker, M. Kirschfink, M. S. Simon, V. Brade, P. F. Zipfel, and R. Wallich. 2003. Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins. J. Biol. Chem. 279:2421-2429.
  17. Kraiczy, P., J. Hellwage, C. Skerka, M. Kirschfink, V. Brade, P. F. Zipfel, and R. Wallich. 2003. Immune evasion of Borrelia burgdorferi: mapping of a complement inhibitor factor H-binding site of BbCRASP-3, a novel member of the Erp protein family. Eur. J. Immunol. 33:697-707.[CrossRef][Medline]
  18. Kraiczy, P., C. Skerka, and P. F. Zipfel. 2001. Further characterization of complement regulator-acquiring surface proteins of Borrelia burgdorferi. Infect. Immun. 69:7800-7809.[Abstract/Free Full Text]
  19. Kwok, S. C., and R. S. Hodges. 2004. Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded alpha helical coiled-coils. J. Biol. Chem. 279:21576-21588.[Abstract/Free Full Text]
  20. Lupas, A., M. Van Dyke, and J. Stock. 1991. Predicting coiled coils from protein sequences. Science 252:1162-1164.[CrossRef][Medline]
  21. Malashkevich, V. N., M. Singh, and P. S. Kim. 2001. The trimer-of-hairpins motif in membrane fusion: Visna virus. Proc. Natl. Acad. Sci. USA 98:8502-8506.[Abstract/Free Full Text]
  22. McDowell, J. V., S. Y. Sung, L. T. Hu, and R. T. Marconi. 2002. Evidence that the variable regions of the central domain of VlsE are antigenic during infection with the Lyme disease spirochetes. Infect. Immun. 70:4196-4203.[Abstract/Free Full Text]
  23. McDowell, J. V., S.-Y. Sung, G. Price, and R. T. Marconi. 2001. Demonstration of the genetic stability and temporal expression of select members of the Lyme disease spirochete OspF protein family during infection in mice. Infect. Immun. 69:4831-4838.[Abstract/Free Full Text]
  24. McDowell, J. V., E. Tran, D. Hamilton, J. Wolfgang, K. Miller, and R. T. Marconi. 2003. Analysis of the ability of spirochete species associated with relapsing fever, avian borreliosis, and epizootic bovine abortion to bind factor H and cleave C3b. J. Clin. Microbiol. 41:3905-3910.[Abstract/Free Full Text]
  25. McDowell, J. V., J. Wolfgang, L. Senty, C. M. Sundy, M. J. Noto, and R. T. Marconi. 2004. Demonstration of the involvement of outer surface protein E coiled-coil structural domains and higher order structural elements in the binding of infection-induced antibody and the complement-regulatory protein, factor H. J. Immunol. 173:7471-7480.[Abstract/Free Full Text]
  26. McDowell, J. V., J. Wolfgang, E. Tran, M. S. Metts, D. Hamilton, and R. T. Marconi. 2003. Comprehensive analysis of the factor H binding capabilities of Borrelia species associated with Lyme disease: delineation of two distinct classes of factor H binding proteins. Infect. Immun. 71:3597-3602.[Abstract/Free Full Text]
  27. Meri, T., A. Hartmann, D. Lenk, R. Eck, R. Wurzner, J. Hellwage, S. Meri, and P. F. Zipfel. 2002. The yeast Candida albicans binds complement regulators factor H and FHL-1. Infect. Immun. 70:5185-5192.[Abstract/Free Full Text]
  28. Meri, T., T. S. Jokiranta, J. Hellwage, A. Bialonski, P. F. Zipfel, and S. Meri. 2002. Onchocerca volvulus microfilariae avoid complement attack by direct binding of factor H. J. Infect. Dis. 185:1786-1793.[CrossRef][Medline]
  29. Metts, S., J. V. McDowell, M. Theisen, P. R. Hansen, and R. T. Marconi. 2003. Analysis of the OspE determinants involved in the binding of factor H and OspE targeting antibodies elicited during infection in mice. Infect. Immun. 71:3587-3596.[Abstract/Free Full Text]
  30. Nguyen, T.-P. K., T. T. Lam, S. W. Barthold, S. R. Telford III, R. A. Flavell, and E. Fikrig. 1994. Partial destruction of Borrelia burgdorferi within ticks that engorged on OspE- or OspF-immunized mice. Infect. Immun. 62:2079-2084.[Abstract/Free Full Text]
  31. Ram, S., D. P. McQuillen, S. Gulati, C. Elkins, M. K. Pangburn, and P. A. Rice. 1998. Binding of complement factor H to loop 5 of porin protein 1A: a molecular mechanism of serum resistance of nonsialyated Neiserria gonorrhoeae. J. Exp. Med. 188:671-680.[Abstract/Free Full Text]
  32. Roberts, D., M. Caimano, J. McDowell, M. Theisen, A. Holm, S. Alban, E. Orff, D. Nelson, S. Wikel, J. Radolf, and R. Marconi. 2002. Environmental regulation and differential expression of members of the Bdr protein family of Borrelia burgdorferi. Infect. Immun. 70:7033-7041.[Abstract/Free Full Text]
  33. Sung, S. Y., J. McDowell, J. A. Carlyon, and R. T. Marconi. 2000. Mutation and recombination in the upstream homology box-flanked ospE-related genes of the Lyme disease spirochetes result in the development of new antigenic variants during infection. Infect. Immun. 68:1319-1327.[Abstract/Free Full Text]
  34. Sung, S. Y., J. V. McDowell, and R. T. Marconi. 2001. Evidence for the contribution of point mutations to vlsE variation and for apparent constraints on the net accumulation of sequence changes in vlsE during infection with the Lyme disease spirochetes. J. Bacteriol. 168:5855-5861.
  35. Whaley, K., and S. Ruddy. 1976. Modulation of the alternative complement pathways by beta 1 H globulin. J. Exp. Med. 144:1147-1163.[Abstract/Free Full Text]
  36. Zhang, J.-R., J. M. Hardham, A. G. Barbour, and A. G. Norris. 1997. Antigenic variation in Lyme disease borreliae by promiscuous recombination of vmp like sequence cassettes. Cell 89:275-285.[CrossRef][Medline]
  37. Zhang, J. R., and S. J. Norris. 1998. Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion. Infect. Immun. 66:3698-3704.[Abstract/Free Full Text]
  38. Zipfel, P. F., C. Skerka, J. Hellwage, S. T. Jokiranta, S. Meri, V. Brade, P. Kraiczy, M. Noris, and G. Remuzzi. 2002. Structure-function studies of the complement system. Biochem. Soc. Trans. 30:971-978.[CrossRef][Medline]

Journal of Bacteriology, February 2005, p. 1317-1323, Vol. 187, No. 4
0021-9193/05/$08.00+0     doi:10.1128/JB.187.4.1317-1323.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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