Pathogenic bacteria from the Neisseriaceae and Pasteurellacea families acquire iron directly from the host iron-binding glycoprotein, transferrin (Tf), in a process mediated by surface receptor proteins that directly bind host Tf, extract the iron, and transport it across the outer membrane. The bacterial Tf receptor is comprised of a surface exposed lipoprotein, Tf-binding protein B (TbpB), and an integral outer-membrane protein, Tf-binding protein A (TbpA), both of which are essential for survival in the host. In this study, we report the 1.98 A resolution structure of TbpB from the porcine pathogen Actinobacillus pleuropneumoniae, providing insights into the mechanism of Tf binding and the role of TbpB. A model for the complex of TbpB bound to Tf is proposed. Mutation of a single surface-exposed Phe residue on TbpB within the predicted interface completely abolishes binding to Tf, suggesting that the TbpB N lobe comprises the sole high-affinity binding region for Tf.

Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin (Tf) during infection via a surface Tf receptor system composed of proteins TbpA and TbpB. Here in we present the crystal structures of TbpB from N. meningitidis, in its apo form and in complex with human Tf (hTf). The structure reveals how TbpB sequesters hTf and initiates iron release from hTf.

A common adaptation among several highly host-adapted Gram-negative species from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families that exclusively reside in the upper respiratory tract is the ability to directly bind host transferrin (Tf) and use it as a source of iron for growth (1, 2). Iron-loaded transferrin is captured by surface receptors that remove iron from Tf and transport the iron across the outer membrane, where it is subsequently transported into the cell through a periplasm binding protein-dependent ABC (ATP binding cassette) transport system. Early observations that the interaction of the bacterial receptors with Tf was highly host specific (3-5) provided a rational explanation for the strict host specificity of these bacterial pathogens.The initial capture of iron-loaded Tf is mediated by a surface lipoprotein, Tf binding protein B (TbpB), which consists of two structurally equivalent lobes preceded by a relatively long anchoring peptide that would allow the protein to extend far from the outer membrane surface (6). The role of TbpB is to capture the iron-loaded form of Tf and deliver it to Tf binding protein A (TbpA), the integral outer membrane protein that serves as the channel for transporting iron across the outer membrane. The structure of a Tf-TbpB complex has recently been determined (7), revealing that that the process of binding iron-loaded Tf does not involve substantial changes in the conformation of the TbpB Nlobe or the Tf C-lobe and effectively traps the Tf C-lobe in the iron-loaded state. In contrast, binding of Tf to TbpA involves substantial conformational changes in the TbpB C-lobe, resulting in substantial separation of the C1 and C2 domains that both contribute ligands for coordination of iron (8). In the absence of structural information for TbpA alone one can only speculate on the conformational changes that occur in the surface loop structures of TbpA upon binding Tf.The process by which TbpB mediates the initial capture of iron-loaded Tf and transfers it to TbpA is only partly understood. The variable association of the anchoring peptide with the C-lobe (9) and its requirement for formation of the ternary complex (10) may indicate that modulation of the anchor peptide may be involved. Although structural models can be developed for the ternary complex (2, 8), these are not based on high-resolution structural information for the actual complex, and how TbpB maintains an interaction with Tf upon domain separation is still not resolved. Similarly, the process by which iron is released and transported across the outer membrane and the degree to which different regions of TbpB participate in this process is uncertain.Since the first discovery of the bacterial Tf receptors (11, 12) and the demonstration of their exquisite host specificity (4), they were postulated to be essential for survival in the native host and thus potentially ideal vaccine targets. The importance of the receptor proteins has been confirmed in a male gonococcal infection

Alignment of amino‐acid sequences from the N‐terminal and C‐terminal halves of transferrin‐binding protein B revealed an underlying bilobed nature with several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose‐binding protein (Mbp) with intact TbpB, its N‐terminal half or C‐terminal half from the human pathogens Neisseria meningitidis and Moraxella catarrhalis were produced. Solid‐phase binding assays and affinity isolation assays demonstrated that the N‐terminal and C‐terminal halves of TbpB could bind independently to human transferrin (hTf). A solid‐phase overlapping synthetic peptide library representing the amino‐acid sequence of hTf was probed with soluble, labelled Mbp–TbpB fusions to localize TbpB‐binding regions on hTf. An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from both organisms, demonstrating a conserved TbpB–hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which included 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 peptide‐binding regions on a molecular model of hTf revealed a series of nearly adjacent surface regions that nearly encircled each lobe. Binding studies with chimeric hTf/bTf transferrins demonstrated that regions in the C‐lobe of hTf were preferentially recognized by the N‐terminal half of TbpB. Collectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf‐binding regions.

Iron-saturated human transferrin was digested with either chymotrypsin or trypsin to produce C-lobe and N-lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C-lobe and N-lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C-lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transferrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin-binding proteins (Tbps), we demonstrated that both transferrin-binding proteins were able to bind to the C-lobe fragment of human transferrin.