Search

Neisseria comprise a large family of Gram-negative bacteria that colonize humans. Two family members, Neisseria gonorrhoeae and Neisseria meningitidis, are pathogens that invade the urogenital tract and nasopharynx, respectively, causing [...], Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co- receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.

Research has been conducted on the human transferrins. The mechanisms of iron release from transferrin in cells and in vitro and of the synergistic iron binding have been investigated and are discussed.

Research has been conducted on the eukaryotic transferrins, bilobal iron-binding proteins with the binding site in each lobe. The separation of the lobes after the full-length protein expression has been carried out and the results demonstrate the the primary receptor-recognition transferrin site is in its C-lobe.

An investigation was conducted to determine the formal redox potential of the Fe in FeCTf, FeNTf and Fe2Tf at endosomal pH of 5.8. An optically transparent thin-layer electrochemical cell and a spectroelectrochemical technique were utilized. Results showed the redox properties of two sites are similar and the redox potentials are too negative for a reductive mechanism to be involved in the endosomal release of Fe from transferrin without coupling to a cascade of reactions which include chelation of Fe.

The process of release of iron from transferrin, an iron-transporting protein, involves remote amino acid residues. Fluoresceinated transferrin studies indicated that Lys 569 is a kinetically active site in the C-terminal protein lobe. The Role of Lys 569 in the release process at pH 5.6, the pH of the endosome where iron transfers from transferrin to iron-dependent cells, was investigated based on site-directed mutagenesis. Release mechanisms were found to have major differences in the two lobes of transferrin.

The rate of iron release from the H249Y N-lobe mutant of human transferrin is much higher than the release from other mutants such as R124S, R124K, K206R, H207E, and Y95H. Iron release from the N-lobe K206 and H207 mutants is very slow compared to the iron release from wild-type N-lobe. The mutation of R124 accentuates iron release both at the endosomal pH of 5.6 and at extracellular pH of 7.4. A simple model that accounts for the negative effect of N-lobe iron release is presented.

Ionic force influences the speed at which iron is released from the two sides of free transferrin. A kinetically active site of the proton must bind to a simple nonchelating anion to induce the release of iron. Binding of iron takes place when the synergistic anion-binding site is inhabited. A complicated release of iron is seen by complexing transferrin to its receptor. The different pH values cause the quick and slow release of iron. The monoferric human transferrin releases iron from its C-terminal and is controlled by a spectrofluorometric process.

Cryo-electron microscopy is used to uncover the molecular structure of the formation of the human transferrin receptor-transferrin complex (TfR - Tf). The structure helps understanding the differences in the iron-release properties of free and receptor bound Tf.

Iron-donating cycle of transferrin is examined. Findings suggest that iron complex with the transferrin receptor throughout the transferrin-to-cell cycle of iron uptake raise the reduction potential to more than 200mV.

Hydroxyl radical-medicated protein footprinting and mass spectrometry was used to reveal the conformational changes that occur upon complex formation for the human transferrin C-lobe bound to the ectodomain of human transferrin receptor 1. By using available genetic, biochemical, and structural data, it was confirmed that the conserved RGD sequence in the helical domain of the transferrin receptor is a primary binding site for the transferrin C-lobe.

Human transferrin, like other members of the transferrin class of iron-binding proteins, is a bilobal structure, the product of duplication and fusion of an ancestral gene during the course of biochemical evolution. The possibility of faster release in thereceptor complex of the full length protein at endosomal pH contributes to the evolutionary advantage of the bilobal structure is considered.