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N-lobe versus C-lobe complexation of bismuth by human transferrin.

ABSTRACT: Interactions of recombinant N-lobe of human serum transferrin (hTF/2N) with Bi3+, a metal ion widely used in medicine, have been investigated by both UV and NMR spectroscopy. The bicarbonate-independent stability constant for Bi3+ binding (K*) to hTF/2N was determined to be log K* 18.9+/-0.2 in 5 mM bicarbonate/10 mM Hepes buffer at 310 K, pH7.4. The presence of Fe3+ in the C-lobe of intact hTF perturbed Bi3+ binding to the N-lobe, whereas binding of Bi3+ to the C-lobe was unaffected by the presence of Fe3+ in the N-lobe. Reactions of Bi3+ (as bismuth nitrilotriacetate or ranitidine bismuth citrate) with hTF/2N in solutions containing 10 mM bicarbonate induced specific changes to high-field 1H-NMR peaks. The 1H co-ordination shifts induced by Bi3+ were similar to those induced by Fe3+ and Ga3+, suggesting that Bi3+ binding causes similar structural changes to those induced by hTF/2N. 13C-NMR data showed that carbonate binds to hTF/2N concomitantly with Bi3+.

SUBMITTER: Sun H

PROVIDER: S-EPMC1219942 | biostudies-other | 1999 Jan

REPOSITORIES: biostudies-other

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Iron and bismuth bound human serum transferrin reveals a partially-opened conformation in the N-lobe.

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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.

Project description: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.

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Structural and functional consequences of the substitution of glycine 65 with arginine in the N-lobe of human transferrin.

Project description:The G65R mutation in the N-lobe of human transferrin was created to mimic a naturally occurring variant (G394R) found in the homologous C-lobe. Because Gly65 is hydrogen-bonded to the iron-binding ligand Asp63, it comprises part of the second-shell hydrogen bond network surrounding the iron within the metal-binding cleft of the protein. Substitution with an arginine residue at this position disrupts the network, resulting in much more facile removal of iron from the G65R mutant. As shown by UV-vis and EPR spectroscopy, and by kinetic assays measuring the release of iron, the G65R mutant can exist in three forms. Two of the forms (yellow and pink in color) are interconvertible. The yellow form predominates in 1 M bicarbonate; the pink form is generated from the yellow form upon exchange into 1 M HEPES buffer (pH 7.4). The third form (also pink in color) is produced by the addition of Fe(3+)-(nitrilotriacetate)(2) to apo-G65R. Hydrogen-deuterium exchange experiments are consistent with all forms of the G65R mutant assuming a more open conformation. Additionally, mass spectrometric analysis reveals the presence of nitrilotriacetate in the third form. The inability to obtain crystals of the G65R mutant led to development of a novel crystallization strategy in which the G65R/K206E double mutation stabilizes a single closed pink conformer and captures Arg65 in a single position. Collectively, these studies highlight the importance of the hydrogen bond network in the cleft, as well as the inherent flexibility of the N-lobe which, although able to adapt to accommodate the large arginine substitution, exists in multiple conformations.

| S-EPMC2693239 | biostudies-literature

A loop in the N-lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping.

Project description:Transferrin (TF) is a bilobal transport protein that acquires ferric iron from the diet and holds it tightly within the cleft of each lobe (thereby preventing its hydrolysis). The iron is delivered to actively dividing cells by receptor mediated endocytosis in which diferric TF preferentially binds to TF receptors (TFRs) on the cell surface and the entire complex is taken into an acidic endosome. A combination of lower pH, a chelator, inorganic anions, and the TFR leads to the efficient release of iron from each lobe. Identification of residues/regions within both TF and TFR required for high affinity binding has been an ongoing goal in the field. In the current study, we created human TF (hTF) mutants to identify a region critical to the interaction with the TFR which also constitutes part of an overlapping epitope for two monoclonal antibodies (mAbs) to the N-lobe, one of which was previously shown to block binding of hTF to the TFR. Four single point mutants, P142A, R143A, K144A, and P145A in the N-lobe, were placed into diferric hTF. Isothermal titration calorimetry (ITC) revealed that three of the four residues (Pro142, Lys144, and Pro145) in this loop are essential to TFR binding. Additionally, Lys144 is common to the recognition of both mAbs which show different sensitivities to the three other residues. Taken together these studies prove that this loop is required for binding of the N-lobe of hTF to the TFR, provide a more precise description of the role of each residue in the loop in the interaction with the TFR, and confirm that the N-lobe is essential to high affinity binding of diferric hTF to TFR.

| S-EPMC4479294 | biostudies-literature

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