Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis

ABSTRACT A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

[1]  P. Dumas,et al.  Evaluation of transferrin-binding protein 2 within the transferrin-binding protein complex as a potential antigen for future meningococcal vaccines , 1995, Infection and immunity.

[2]  G. Holdgate.  Making cool drugs hot: isothermal titration calorimetry as a tool to study binding energetics. , 2001, BioTechniques.

[3]  S. Gray-Owen,et al.  Bacterial transferrin and lactoferrin receptors. , 1996, Trends in microbiology.

[4]  David Eisenberg,et al.  The helical hydrophobic moment: a measure of the amphiphilicity of a helix , 1982, Nature.

[5]  G. Renauld-Mongénie,et al.  Both the full-length and the N-terminal domain of the meningococcal transferrin-binding protein B discriminate between human iron-loaded and apo-transferrin. , 1998, FEMS microbiology letters.

[6]  S. Salzberg,et al.  Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. , 2000, Science.

[7]  A. Schryvers,et al.  Identification and characterization of the transferrin receptor from Neisseria meningitidis , 1988, Molecular microbiology.

[8]  P. Dumas,et al.  Transferrin-binding proteins isolated from Neisseria meningitidis elicit protective and bactericidal antibodies in laboratory animals. , 1993, Vaccine.

[9]  E. Jacobs,et al.  Cloning and characterization of Neisseria meningitidis genes encoding the transferrin-binding proteins Tbp1 and Tbp2. , 1993, Gene.

[10]  D. Caugant,et al.  Intercontinental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic disease. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[11]  P. Sparling,et al.  Characterization of the diversity and the transferrin-binding domain of gonococcal transferrin-binding protein 2 , 1997, Infection and immunity.

[12]  A. Schryvers,et al.  Peptide-Peptide Interactions between Human Transferrin and Transferrin-Binding Protein B from Moraxellacatarrhalis , 2003, Journal of bacteriology.

[13]  N. C. Price,et al.  Phosphoglycerate mutase from Schizosaccharomyces pombe: development of an expression system and characterisation of three histidine mutants of the enzyme. , 1996, Biochimica et biophysica acta.

[14]  I. Stojiljković,et al.  Iron acquisition systems in the pathogenic Neisseria , 1999, Molecular microbiology.

[15]  A. Schryvers,et al.  Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin‐binding protein B , 1999, Molecular microbiology.

[16]  J. W. Taylor,et al.  Structure-function analysis of proteins through the design, synthesis, and study of peptide models. , 1987, Methods in enzymology.

[17]  D. Caugant,et al.  Allelic Diversity of the Two Transferrin Binding Protein B Gene Isotypes among a Collection of Neisseria meningitidisStrains Representative of Serogroup B Disease: Implication for the Composition of a Recombinant TbpB-Based Vaccine , 2000, Infection and Immunity.

[18]  E. Jacobs,et al.  Characterization of genetic exchanges between various highly divergent tbpBs, having occurred in Neisseria meningitidis. , 1998, Gene.

[19]  B. Rokbi,et al.  Diversity of the transferrin-binding protein Tbp2 of Neisseria meningitidis. , 1995, Gene.

[20]  A. Jerse,et al.  Gonococcal Genes Encoding Transferrin-Binding Proteins A and B Are Arranged in a Bicistronic Operon but Are Subject to Differential Expression , 2001, Infection and Immunity.

[21]  A. Thomas,et al.  A fast method to predict protein interaction sites from sequences. , 2000, Journal of molecular biology.

[22]  E. Jacobs,et al.  Characterization of a highly structured domain in Tbp2 from Neisseria meningitidis involved in binding to human transferrin , 1994, Journal of bacteriology.

[23]  E. Jacobs,et al.  Molecular characterization of hybrid Tbp2 proteins from Neisseria meningitides , 1996, Molecular microbiology.

[24]  H De Loof,et al.  Use of hydrophobicity profiles to predict receptor binding domains on apolipoprotein E and the low density lipoprotein apolipoprotein B-E receptor. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[25]  A. Schryvers,et al.  Biochemical evidence for a conserved interaction between bacterial transferrin binding protein A and transferrin binding protein B. , 1998, Microbial pathogenesis.

[26]  C Combet,et al.  NPS@: network protein sequence analysis. , 2000, Trends in biochemical sciences.

[27]  B. Rokbi,et al.  Identification of two major families of transferrin receptors among Neisseria meningitidis strains based on antigenic and genomic features. , 1993, FEMS microbiology letters.

[28]  D. Caugant,et al.  Evaluation of recombinant transferrin-binding protein B variants from Neisseria meningitidis for their ability to induce cross-reactive and bactericidal antibodies against a genetically diverse collection of serogroup B strains , 1997, Infection and immunity.

[29]  D. Eisenberg,et al.  Analysis of membrane and surface protein sequences with the hydrophobic moment plot. , 1984, Journal of molecular biology.

[30]  R. W. Evans,et al.  Characterisation of the meningococcal transferrin binding protein complex by photon correlation spectroscopy , 1997, FEBS letters.

[31]  R. Brasseur,et al.  Differential functionalities of amphiphilic peptide segments of the cell‐septation penicillin‐binding protein 3 of Escherichia coli , 2000, Molecular microbiology.

[32]  Andrew Gorringe,et al.  Insight into the Structure and Function of the Transferrin Receptor from Neisseria meningitidis Using Microcalorimetric Techniques* , 2003, The Journal of Biological Chemistry.

[33]  G. Renauld-Mongénie,et al.  Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B , 1997, Journal of bacteriology.