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Purified meningococcal transferrin-binding protein B interacts with a secondary, strain-specific, binding site in the N-terminal lobe of human transferrin.

ABSTRACT: Neisseria meningitidis, grown in iron-limited conditions, produces two transferrin-binding proteins (TbpA and TbpB) that independently and specifically bind human serum transferrin (hTF) but not bovine serum transferrin (bTF). We have used surface plasmon resonance to characterize the interaction between individual TbpA and TbpB and a series of full-length human-bovine chimaeric transferrins (hbTFs) under conditions of variable saturation with iron. A comparative analysis of hTF and hbTF chimaera-binding data confirmed that the major features involved in Tbp binding are located in the C-terminal lobe of hTF and that isolated TbpA can recognize distinct sites present in, or conformationally influenced by, residues 598-679. Binding by TbpB was maintained at a significant but decreased level after replacement of the entire hTF C-terminal lobe by the equivalent bovine sequence. The extent of this binding difference was dependent on the meningococcal strain and on the presence of hTF residues 255-350. This indicated that TbpB from strain SD has a secondary, strain-specific, binding site located within this region, whereas TbpB from strain B16B6 does not share this recognition site. Binding of TbpA was influenced primarily by sequence substitutions in the hTF C-terminal lobe, and co-purified TbpA and TbpB (TbpA+B) was functionally distinct from either of its components. The limited divergence between hTF and bTF has been related to observed differences in binding by Tbps and has been used to delineate those regions of hTF that are important for such interactions.

SUBMITTER: Boulton IC

PROVIDER: S-EPMC1220138 | biostudies-other | 1999 Apr

REPOSITORIES: biostudies-other

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Project description:Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process. To identify hTf binding sites within the meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays. Both the N-terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesis approach, two and five hTf binding sites were identified in the N- and C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 394) in the ligand interaction. Taken together, the results indicated that the N- and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain. Antisera raised to both N- and C-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the N- and C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.

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Purified meningococcal transferrin-binding protein B interacts with a secondary, strain-specific, binding site in the N-terminal lobe of human transferrin.

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