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.

FIG. 1.

Plots of <μ_H> (10⁻¹ kcal/mol) versus <H> (10⁻¹ kcal/mol) of residues ⁴²SGAAYGFAVKLPRRNAHFNP⁶¹ (annotated residues, K51 to A57) of TbpB of N. meningitidis strain B16B6. (A) Residues ⁵³PRRNA⁵⁷ were found to lie within the RBD zone. (B to D) Evaluation of effects of single-amino-acid replacements on the positions of the residues in the RDB zone, indicating changes in binding potential. K51N has no significant effect on the RBD zone (B); R54N (C) and R55N (D) abolish the binding potential (the RBD zone is almost empty).

FIG. 2.

Sequence of mature TbpB of N. meningitidis strain B16B6. Amino acids predicted to have a strong (red), moderate (blue), or weak (green) binding potential are highlighted. The three major receptor-binding domains are boxed.

FIG. 3.

Western blot analysis of wild-type and point-mutated MBP-TbpB with HRP-htf. Crude extracts with equivalent amounts of expressed wild-type or mutated MBP-TbpB were analyzed for their reactivities to HRP-hTf at 10 μg/ml.

FIG. 4.

Far-UV CD spectra of wild-type and mutated MBP-TbpB proteins. ———, wild-type MBP-TbpB; ———, R55N; - - - - - - - -, R80N; — - - — - - —, R243N. The CD spectrum of mutant R55E, which for clarity is not shown, is superimposable on these spectra. Control, MBP alone (— · — · —).