Transferrin-binding protein B of Neisseria meningitidis: sequence-based identification of the transferrin-Binding site confirmed by site-directed mutagenesis.
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.
Renauld-Mongénie
Geneviève
G
Aventis Pasteur, 69280 Marcy l'Etoile, France. genevieve.renauld@aventis.com
Lins
Laurence
L
Krell
Tino
T
Laffly
Laure
L
Mignon
Michèle
M
Dupuy
Monique
M
Delrue
Rose-May
RM
Guinet-Morlot
Françoise
F
Brasseur
Robert
R
Lissolo
Ling
L
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D001665:Binding Sites; D002942:Circular Dichroism; D022401:Meningococcal Vaccines / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009345:Neisseria meningitidis / Q000737:chemistry*; D014168:Transferrin / Q000378:metabolism*; D033882:Transferrin-Binding Protein B / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
D000907:Antibodies, Bacterial; D022401:Meningococcal Vaccines; D014168:Transferrin; D033882:Transferrin-Binding Protein B