Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin-binding protein B

Alignment of amino-acid sequences from the N-terminal and C-terminal halves of transferrin-binding protein B revealed an underlying bilobed nature wit h several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose-binding protein (Mbp) with intact TbpB, its N-t erminal half or C-terminal half from the human pathogens Neisseria meningit idis and Moraxella catarrhalis were produced. Solid-phase binding assays an d affinity isolation assays demonstrated that the N-terminal and C-terminal halves of TbpB could bind independently to human transferrin (hTf), A soli d-phase overlapping synthetic peptide library representing the amino-acid s equence of hTf was probed with soluble, labelled Mbp-TbpB fusions to locali ze TbpB-binding regions on hTf, An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from b oth organisms, demonstrating a conserved TbpB-hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which inclu ded peptides from equivalent regions on the two hTf lobes, indicating that TbpB interacts with each lobe of hTf in a similar manner. Mapping of the pe ptide-binding regions on a molecular model of hTf revealed a series of near ly adjacent surface regions that nearly encircled each lobe, Binding studie s with chimeric hTf/bTf transferrins demonstrated that regions in the C-lob e of hTf were preferentially recognized by the N-terminal half of TbpB. Col lectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf-binding regions.