scholarly journals Delineation of Species-Specific Binding Properties of the CspZ Protein (BBH06) of Lyme Disease Spirochetes: Evidence for New Contributions to the Pathogenesis of Borrelia spp.

2007 ◽  
Vol 75 (11) ◽  
pp. 5272-5281 ◽  
Author(s):  
Elizabeth A. Rogers ◽  
Richard T. Marconi
Keyword(s):  
Lyme Disease ◽  
Ligand Binding ◽  
Dna Sequence ◽  
Molecular Basis ◽  
Serum Proteins ◽  
Specific Binding ◽  
Coiled Coil ◽  
Factor H ◽  
Site Directed Mutagenesis ◽  
Binding Properties

ABSTRACT Borrelia burgdorferi CspZ (TIGR open reading frame designation, BBH06) is part of a functionally related group of proteins that bind one or more members of the factor H (FH) protein family. In this report we assess the conservation, distribution, properties, and ligand binding abilities of CspZ from the three main Borrelia species associated with Lyme disease infections in humans. CspZ (also referred to as BbCRASP-2 in the literature) was found to be highly conserved at the intraspecies level but divergent at the interspecies level. All CspZ orthologs that originated from B. burgdorferi isolates bound FH from a diverse group of mammals. In contrast, CspZ derived from B. garinii and B. afzelii did not. Regardless of the Borrelia species of origin, all CspZ proteins tested bound to unknown ∼60-kDa serum proteins produced by different mammals. To further define the molecular basis for the differential binding of CspZ orthologs to host proteins, DNA sequence, truncation, and site-directed mutagenesis analyses were performed. DNA sequence analyses revealed that B. garinii and B. afzelii CspZ orthologs possess a 64-amino-acid N-terminal domain that is absent from B. burgdorferi CspZ. However, binding analyses of recombinant proteins revealed that this domain does not in and of itself influence ligand binding properties. Truncation and mutagenesis analyses further revealed that the key determinants required for ligand binding are discontinuous and that the presentation of the ligand binding pocket is dependent on alpha helices with high coiled-coil formation probability. The data presented here provide insight into the molecular basis of CspZ-ligand interactions and suggest that CspZ orthologs from diverse Borrelia species can contribute to the host-pathogen interaction through their interaction with serum proteins.

scholarly journals Identification of an Antiparallel Coiled-Coil/Loop Domain Required for Ligand Binding by the Borrelia hermsii FhbA Protein: Additional Evidence for the Role of FhbA in the Host-Pathogen Interaction

2008 ◽  
Vol 76 (5) ◽  
pp. 2113-2122 ◽  
Author(s):  
Kelley M. Hovis ◽  
John C. Freedman ◽  
Hongming Zhang ◽  
Jonathan L. Forbes ◽  
Richard T. Marconi
Keyword(s):  
Serum Proteins ◽  
Coiled Coil ◽  
Factor H ◽  
Site Directed Mutagenesis ◽  
Relapsing Fever ◽  
Borrelia Hermsii ◽  
Alpha Helices ◽  
Host Pathogen Interaction ◽  
Loop Domain ◽  
Host Pathogen

ABSTRACT Borrelia hermsii, an etiological agent of tick-borne relapsing fever in North America, binds host-derived serum proteins including factor H (FH), plasminogen, and an unidentified 60-kDa protein via its FhbA protein. Two distinct phylogenetic types of FhbA have been delineated (FhbA1 and FhbA2). These orthologs share a conserved C-terminal domain that contains two alpha helices with a high predictive probability of coiled-coil formation that are separated by a 14-amino-acid loop domain. Through site-directed mutagenesis, we have identified residues within these domains that influence the binding of both mouse and human FH, plasminogen, and/or the 60-kDa protein. To further investigate the involvement of FhbA in the host-pathogen interaction, strains that are either FhbA+ (isolate YOR) or FhbA− (isolate REN) were tested for serum sensitivity. Significant differences were observed, with YOR and REN being serum resistant and serum sensitive (intermediate), respectively. To test the abilities of these strains to infect and persist in mice, mice were needle inoculated, and infectivity and persistence were then assessed. While both strains REN and YOR infected mice, only the FhbA+ YOR strain persisted beyond day 4. Survival of the YOR isolate in blood correlated with the upregulation of the fhbA gene, as demonstrated by real-time reverse transcriptase PCR. These data advance our understanding of the unique interactions of FhbA with individual serum proteins and provide support for the hypothesis that FhbA is an important contributor to the pathogenesis of the relapsing fever spirochete B. hermsii.

scholarly journals Comparative Analysis of the Properties and Ligand Binding Characteristics of CspZ, a Factor H Binding Protein, Derived from Borrelia burgdorferi Isolates of Human Origin

2009 ◽  
Vol 77 (10) ◽  
pp. 4396-4405 ◽  
Author(s):  
Elizabeth A. Rogers ◽  
Shane V. Abdunnur ◽  
John V. McDowell ◽  
Richard T. Marconi
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Ligand Binding ◽  
Serum Proteins ◽  
Early Stage ◽  
Regulatory Protein ◽  
Factor H ◽  
Serum Samples ◽  
Binding Characteristics ◽  
Protein Factor

ABSTRACT Borrelia burgdorferi CspZ (BBH06/BbCRASP-2) binds the complement regulatory protein factor H (FH) and additional unidentified serum proteins. The goals of this study were to assess the ligand binding capability of CspZ orthologs derived from an extensive panel of human Lyme disease isolates and to further define the molecular basis of the interaction between FH and CspZ. While most B. burgdorferi CspZ orthologs analyzed bound FH, specific, naturally occurring polymorphisms, most of which clustered in a specific loop domain of CspZ, prevented FH binding in some orthologs. Sequence analyses also revealed the existence of CspZ phyletic groups that correlate with FH binding and with the relationships inferred from ribosomal spacer types (RSTs). CspZ type 1 (RST1) and type 3 (RST3) strains bind FH, while CspZ type 2 (RST2) strains do not. Antibody responses to CspZ were also assessed. Anti-CspZ antibodies were detected in mice by week 2 of infection, indicating that there was expression during early-stage infection. Analyses of sera collected from infected mice suggested that CspZ production continued over the course of long-term infection as the antibody titer increased over time. While antibody to CspZ was detected in several human Lyme disease serum samples, the response was not universal, and the titers were generally low. Vaccination studies with mice demonstrated that while CspZ is immunogenic, it does not elicit an antibody that is protective or that inhibits dissemination. The data presented here provide significant new insight into the interaction between CspZ and FH and suggest that there is a correlation between CspZ production and dissemination. However, in spite of its possible contributory role in pathogenesis, the immunological analyses indicated that CspZ is likely to have limited potential as a diagnostic marker and vaccine candidate for Lyme disease.

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

2005 ◽  
Vol 187 (4) ◽  
pp. 1317-1323 ◽  
Author(s):  
John V. McDowell ◽  
Matthew E. Harlin ◽  
Elizabeth A. Rogers ◽  
Richard T. Marconi
Keyword(s):  
Lyme Disease ◽  
Binding Site ◽  
Immune Evasion ◽  
Coiled Coil ◽  
Factor H ◽  
Regulatory Proteins ◽  
Structural Elements ◽  
Binding Ability ◽  
Complement Regulatory Proteins ◽  
Factor I

ABSTRACT 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.

scholarly journals Interactions of new lactoglobulin variants with tetracaine: crystallographic studies of ligand binding to lactoglobulin mutants possessing single substitution in the binding pocket

2021 ◽  
Author(s):  
Joanna Loch ◽  
Piotr Bonarek ◽  
Monika Siuda ◽  
Paulina Wróbel ◽  
Krzysztof Lewiński
Keyword(s):  
Ligand Binding ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Anesthetic Drug ◽  
Binding Properties ◽  
X Ray Crystallography ◽  
Local Site ◽  
In Silico Studies ◽  
Β Lactoglobulin ◽  
Local Anesthetic Drug

β-Lactoglobulin (BLG) like other lipocalins can be modified by mutagenesis to re-direct its ligand binding properties. Local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change BLG ligand preferences. The presented studies are focused on previously described mutants L39Y, I56F, L58F, F105L, and M107L and two new BLG variants, L39K and F105A, and their interactions with local anesthetic drug tetracaine. Binding of tetracaine to BLG mutants was investigated by X-ray crystallography. Structural analysis revealed that for tetracaine binding, the shape of the binding pocket seems to be a more important factor than the substitutions influencing the number of interactions. Analyzed BLG mutants can be classified according to their binding properties to variants: capable of binding tetracaine in the β-barrel (L58F, M107L); capable of accommodating tetracaine on the protein surface (I56F) and unable to bind tetracaine (F105L). Variants L39K, L39Y, and F105A, had a binding pocket blocked by endogenous fatty acids. The new tetracaine binding site was found in the I56F variant. The site localized on the surface near Arg124 and Trp19 was previously predicted by in silico studies and was confirmed in the crystal structure.

THE THYROXINE-BINDING PROPERTIES OF SERUM PROTEINS. A COMPETITIVE BINDING TECHNIQUE EMPLOYING SEPHADEX G-25

1975 ◽  
Vol 65 (3) ◽  
pp. 319-332 ◽  
Author(s):  
R. L. SUTHERLAND ◽  
M. W. SIMPSON-MORGAN
Keyword(s):  
Binding Proteins ◽  
Serum Proteins ◽  
Binding Capacity ◽  
Specific Binding ◽  
Competitive Binding ◽  
Association Constants ◽  
Binding Properties ◽  
Rat Serum ◽  
Human Proteins ◽  
Dilute Blood

SUMMARY A competitive binding technique is described for the estimation of the thyroxine (T4)-binding properties of serum proteins in dilute blood serum and lymph. When used in conjunction with an assay for total T4 the following parameters can be estimated: the number of functionally different T4 binding proteins, their individual association constants and binding capacities for T4, the amount of T4 which is bound to each binding species, and the concentration of unbound (free) T4. Both human and sheep serum have three functionally different T4-binding proteins. The association constants for the three human proteins were 9·5 × 109, 1·6 × 108 and 3·1 × 105 1/mol for T4-binding globulin (TBG), T4-binding prealbumin (TBPA) and serum albumin, respectively. The corresponding sheep proteins, TBG, TBP-2 and albumin, had association constants of 8·9 × 109, 1·4 × 108 and 3·5 × 1051/mol. Human TBG had a mean binding capacity of 21·3 μg/100 ml and that of ovine TBG was 12·8 μg/100 ml. The other specific binding proteins (TBPA in man and TBP-2 in sheep) had mean binding capacities of 307 and 359 μg/100 ml respectively. Two functionally different T4-binding proteins were identified in rat serum.

scholarly journals Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis

2009 ◽  
Vol 13 (12) ◽  
pp. 3127-3138 ◽  
Author(s):  
Heather K. Lamb ◽  
Jingshan Ren ◽  
Alison Park ◽  
Christopher Johnson ◽  
Kris Leslie ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Properties

scholarly journals Selective Binding of Borrelia burgdorferi OspE Paralogs to Factor H and Serum Proteins from Diverse Animals: Possible Expansion of the Role of OspE in Lyme Disease Pathogenesis

2006 ◽  
Vol 74 (3) ◽  
pp. 1967-1972 ◽  
Author(s):  
Kelley M. Hovis ◽  
Emily Tran ◽  
Christina M. Sundy ◽  
Eric Buckles ◽  
John V. McDowell ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Host Range ◽  
Serum Proteins ◽  
Factor H ◽  
Disease Pathogenesis ◽  
Selective Binding

ABSTRACT The binding of Borrelia burgdorferi OspE, OspF, and family 163 (Elp) proteins to factor H/factor H-like protein 1 (FHL-1) and other serum proteins from different animals was assessed. OspE paralogs bound factor H and unidentified serum proteins from a subset of animals, while OspF and Elp proteins did not. These data advance our understanding of factor H binding, the host range of the Lyme spirochetes, and the expanding role of OspE in pathogenesis.

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