Biomedical subjects

Anne B Mason

Publications and source records attributed to Anne B Mason.

16 recordsLinked to original sources

PubMed2006

The crystal structure of iron-free human serum transferrin provides insight into inter-lobe communication and receptor binding.

Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-A resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7A by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4 degrees and 49.5 degrees rotations are required to open the N- and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.

Amino Acid Sequence↗

PubMed2006

Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor.

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.

Base Sequence↗

PubMed2005

Composition of pH-sensitive triad in C-lobe of human serum transferrin. Comparison to sequences of ovotransferrin and lactoferrin provides insight into functional differences in iron release.

The transferrins (TF) are a family of bilobal glycoproteins that tightly bind ferric iron. Each of the homologous N- and C-lobes contains a single iron-binding site situated in a deep cleft. Human serum transferrin (hTF) serves as the iron transport protein in the blood; circulating transferrin binds to receptors on the cell surface, and the complex is internalized by endocytosis. Within the cell, a reduction in pH leads to iron release from hTF in a receptor-dependent process resulting in a large conformational change in each lobe. In the hTF N-lobe, two critical lysines facilitate this pH-dependent conformational change allowing entry of a chelator to capture the iron. In the C-lobe, the lysine pair is replaced by a triad of residues: Lys534, Arg632, and Asp634. Previous studies show that mutation of any of these triad residues to alanine results in significant retardation of iron release at both pH 7.4 and pH 5.6. In the present work, the role of the three residues is probed further by conversion to the residues observed at the equivalent positions in ovotransferrin (Q-K-L) and human lactoferrin (K-N-N) as well as a triad with an interchanged lysine and arginine (K534R/R632K). As expected, all of the constructs bind iron and associate with the receptor with nearly the same K(D) as the wild-type monoferric hTF control. However, interesting differences in the effect of the substitutions on the iron release rate in the presence and absence of the receptor at pH 5.6 are observed. Additionally, titration with KCl indicates that position 632 must have a positively charged residue to elicit a robust rate acceleration as a function of increasing salt. On the basis of these observations, a model for iron release from the hTF C-lobe is proposed. These studies provide insight into the importance of charge and geometry of the amino acids at these positions as a partial explanation for differences in behavior of individual TF family members, human serum transferrin, ovotransferrin, and lactoferrin. The studies collectively highlight important features common to both the N- and C-lobes of TF and the critical role of the receptor in iron release.

Amino Acid Sequence↗

PubMed2005

Mutational analysis of C-lobe ligands of human serum transferrin: insights into the mechanism of iron release.

Each homologous lobe of human serum transferrin (hTF) has one Fe(3+) ion bound by an aspartic acid, a histidine, two tyrosine residues, and two oxygens from the synergistic anion, carbonate. Extensive characterization of these ligands in the N-terminal lobe has been carried out. Despite sharing the same set of ligands, there is a substantial amount of evidence that the N- and C-lobes are inequivalent. Studies of full-length hTF have shown that iron release from each lobe is kinetically distinguishable. To simplify the assessment of mutations in the C-lobe, we have created mutant hTF molecules in which the N-lobe binds iron with high affinity or not at all. Mutations targeting the C-lobe liganding residues have been introduced into these hTF constructs. UV-visible spectral, kinetic, and EPR studies have been undertaken to assess the effects of each mutation and to allow direct comparison to the N-lobe. As found for the N-lobe, the presence of Y517 in the C-lobe (equivalent to Y188 in the N-lobe) is absolutely essential for the binding of iron. Unlike the N-lobe, however, mutation of Y426 (equivalent to Y95) does not produce a stable complex with iron. For the mutants that retain the ability to bind iron (D392S and H585A), the rates of release are considerably slower than those measured for equivalent mutations in the N-lobe at both pH 7.4 and pH 5.6. Equilibrium binding experiments with HeLa S(3) cells indicate that recombinant hTF, in which Y426 or H585 is mutated, favor a closed or nearly closed conformation while those with mutations of the D392 or Y517 ligands appear to promote an open conformation. The differences in the effects of mutating the liganding residues in the two lobes and the subtle indications of cooperativity between lobes point to the importance of the transferrin receptor in effecting iron release from the C-lobe. Significantly, the equilibrium binding experiments also indicate that, regardless of which lobe contains the iron, the free energy of binding is equivalent and not additive; each monoferric hTF has a free energy of binding that is 82% of diferric hTF.

Animals↗

PubMed2005

Evolution of the transferrin family: conservation of residues associated with iron and anion binding.

The transferrin family spans both vertebrates and invertebrates. It includes serum transferrin, ovotransferrin, lactoferrin, melanotransferrin, inhibitor of carbonic anhydrase, saxiphilin, the major yolk protein in sea urchins, the crayfish protein, pacifastin, and a protein from green algae. Most (but not all) contain two domains of around 340 residues, thought to have evolved from an ancient duplication event. For serum transferrin, ovotransferrin and lactoferrin each of the duplicated lobes binds one atom of Fe (III) and one carbonate anion. With a few notable exceptions each iron atom is coordinated to four conserved amino acid residues: an aspartic acid, two tyrosines, and a histidine, while anion binding is associated with an arginine and a threonine in close proximity. These six residues in each lobe were examined for their evolutionary conservation in the homologous N- and C-lobes of 82 complete transferrin sequences from 61 different species. Of the ligands in the N-lobe, the histidine ligand shows the most variability in sequence. Also, of note, four of the twelve insect transferrins have glutamic acid substituted for aspartic acid in the N-lobe (as seen in the bacterial ferric binding proteins). In addition, there is a wide spread substitution of lysine for the anion binding arginine in the N-lobe in many organisms including all of the fish, the sea squirt and many of the unusual family members i.e., saxiphilin and the green alga protein. It is hoped that this short analysis will provide the impetus to establish the true function of some of the TF family members that clearly lack the ability to bind iron in one or both lobes and additionally clarify the evolutionary history of this important family of proteins.

Animals↗

PubMed2005

The molecular mechanism for receptor-stimulated iron release from the plasma iron transport protein transferrin.

Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/F760A-TfR, changes the Tf iron binding site > or =30 A from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits long-range conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.

Antigens, CD↗

PubMed2005

Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor.

The molecular basis of the transferrin (TF)-transferrin receptor (TFR) interaction is not known. The C-lobe of TF is required to facilitate binding to the TFR and both the N- and C-lobes are necessary for maximal binding. Several mAb have been raised against human transferrin (hTF). One of these, designated F11, is specific to the C-lobe of hTF and does not recognize mouse or pig TF. Furthermore, mAb F11 inhibits the binding of TF to TFR on HeLa cells. To map the epitope for mAb F11, constructs spanning various regions of hTF were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The recombinant fusion proteins were analysed in an iterative fashion by immunoblotting using mAb F11 as the probe. This process resulted in the localization of the F11 epitope to the C1 domain (residues 365-401) of hTF. Subsequent computer modelling suggested that the epitope is probably restricted to a surface patch of hTF consisting of residues 365-385. Mutagenesis of the F11 epitope of hTF to the sequence of either mouse or pig TF confirmed the identity of the epitope as immunoreactivity was diminished or lost. In agreement with other studies, these epitope mapping studies support a role for residues in the C1 domain of hTF in receptor binding.

Antibodies, Monoclonal↗

PubMed2004

The low pKa value of iron-binding ligand Tyr188 and its implication in iron release and anion binding of human transferrin.

2D NMR-pH titrations were used to determine pKa values for four conserved tyrosine residues, Tyr45, Tyr85, Tyr96 and Tyr188, in human transferrin. The low pKa of Tyr188 is due to the fact that the iron-binding ligand interacts with Lys206 in open-form and with Lys296 in the closed-form of the protein. Our current results also confirm the anion binding of sulfate and arsenate to transferrin and further suggest that Tyr188 is the actual binding site for the anions in solution. These data indicate that Tyr188 is a critical residue not only for iron binding but also for chelator binding and iron release in transferrin.

Anions↗

PubMed2004

The oxalate effect on release of iron from human serum transferrin explained.

A unique feature of the mechanism of iron binding to the transferrin (TF) family is the synergistic relationship between metal binding and anion binding. Little or no iron will bind to the protein without concomitant binding of an anion, physiologically identified as carbonate. Substitution of oxalate for carbonate produces no significant changes in polypeptide folding or domain orientation in the N-lobe of human serum TF (hTF) as revealed by our 1.2A structure. The oxalate is able to bind to the iron in a symmetric bidentate fashion, which, combined with the low pK(a) of the oxalate anion, makes iron displacement more difficult as documented by both iron release kinetic and equilibrium data. Characterization of an N-lobe in which the arginine at position 124 is mutated to alanine reveals that the stabilizing effect of oxalate is even greater in this mutant and nearly cancels the destabilizing effect of the mutation. Importantly, incorporation of oxalate as the synergistic anion appears to completely inhibit removal of iron from recombinant full-length hTF by HeLa S(3) cells, strongly indicating that oxalate also replaces carbonate in the C-lobe to form a stable complex. Kinetic studies confirm this claim. The combination of structural and functional data provides a coherent delineation of the effect of oxalate binding on hTF and rationalizes the results of many previous studies. In the context of iron uptake by cells, substitution of carbonate by oxalate effectively locks the iron into each lobe of hTF, thereby interfering with normal iron metabolism.

Binding Sites↗

PubMed2004

Indirect detection of protein-metal binding: interaction of serum transferrin with In3+ and Bi3+.

Transferrins comprise a class of monomeric glycoproteins found in all vertebrates, whose function is iron sequestration and transport. In addition to iron, serum transferrin also binds a variety of other metals and is believed to provide a route for the in vivo delivery of such metals to cells. In the present study, ESI MS is used to investigate interactions between human serum transferrin and two nonferrous metals, indium (a commonly used imaging agent) and bismuth (a component of many antiulcer drugs). While the UV-Vis absorption spectroscopy measurements clearly indicate that both metals bind strongly to transferrin in solution, the metal-protein complex can be detected by ESI MS only for indium, but not for bismuth. Despite the apparently low stability of the transferrin-bismuth complex in the gas phase, presence of such complex in solution can be established by ESI MS indirectly. This is done by monitoring the evolution of charge state distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the absence of the metal in solution. The anomalous instability of the transferrin-bismuth complex in the gas phase is rationalized in terms of conformational differences between this form of transferrin and the holo-forms of this protein produced by binding of metals with smaller ionic radii (e.g., Fe3+ and In3+). The large size of Bi3+ ion is likely to prevent formation of a closed conformation (canonical structure of the holo-protein), resulting in a non-native metal coordination. It is suggested that transferrin retains the open conformation (characteristic of the apo-form) upon binding Bi3+, with only two ligands in the metal coordination sphere provided by the protein itself. This suggestion is corroborated by the results of circular dichroism measurements in the near-UV range. Since the cellular consumption of metals in the transferrin cycle critically depends upon recognition of the holo-protein complex by the transferrin receptor, the noncanonical conformation of the transferrin-bismuth complex may explain very inefficient delivery of bismuth to cells even when a high dosage of bismuth-containing drugs is administered for prolonged periods of time.

Bismuth↗

PubMed2004

Expression, purification, and characterization of authentic monoferric and apo-human serum transferrins.

Transferrin is a bilobal protein with the ability to bind iron in two binding sites situated at the bottom of a cleft in each lobe. We have previously described the production of recombinant non-glycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa-His tag at the amino-terminus. Constructs in this background that contain strategic mutations to completely prevent iron binding in each lobe or in both lobes have now been produced. These monoferric hTFs will allow dissection of the contribution of each lobe to transferrin function. In addition, the construct completely lacking in the ability to bind iron in either lobe provides an opportunity to assess whether hTF has any other functions in addition to iron transport. Following insertion of the His-tagged hTF molecules into the pNUT vector, transfection into baby hamster kidney cells and selection with methotrexate, the secreted recombinant proteins were isolated from the tissue culture medium and characterized with regard to their iron binding properties. Significant improvements over our previous protocol include: (1) addition of butyric acid at a level of 1mM which leads to a substantial increase in protein production (as much as a 65% increase compared to control cells); and (2) elimination of an anion exchange column prior to isolation on a Qiagen Ni-NTA column which makes purification of the His-tagged constructs faster and therefore more efficient. These improvements should be applicable to expression of other recombinant proteins in mammalian cells.

Animals↗

PubMed2003

Structural and functional consequences of binding site mutations in transferrin: crystal structures of the Asp63Glu and Arg124Ala mutants of the N-lobe of human transferrin.

Human transferrin is a serum protein whose function is to bind Fe(3+) with very high affinity and transport it to cells, for delivery by receptor-mediated endocytosis. Structurally, the transferrin molecule is folded into two globular lobes, representing its N-terminal and C-terminal halves, with each lobe possessing a high-affinity iron binding site, in a cleft between two domains. Central to function is a highly conserved set of iron ligands, including an aspartate residue (Asp63 in the N-lobe) that also hydrogen bonds between the two domains and an arginine residue (Arg124 in the N-lobe) that binds an iron-bound carbonate ion. To further probe the roles of these residues, we have determined the crystal structures of the D63E and R124A mutants of the N-terminal half-molecule of human transferrin. The structure of the D63E mutant, determined at 1.9 A resolution (R = 0.245, R(free) = 0.261), showed that the carboxyl group still binds to iron despite the larger size of the Glu side chain, with some slight rearrangement of the first turn of alpha-helix residues 63-72, to which it is attached. The structure of the R124A mutant, determined at 2.4 A resolution (R = 0.219, R(free) = 0.288), shows that the loss of the arginine side chain results in a 0.3 A displacement of the carbonate ion, and an accompanying movement of the iron atom. In both mutants, the iron coordination is changed slightly, the principal change being in each case a lengthening of the Fe-N(His249) bond. Both mutants also release iron more readily than the wild type, kinetically and in terms of acid lability of iron binding. We attribute this to more facile protonation of the synergistically bound carbonate ion, in the case of R124A, and to strain resulting from the accommodation of the larger Glu side chain, in the case of D63E. In both cases, the weakened Fe-N(His) bond may also contribute, consistent with protonation of the His ligand being an early intermediate step in iron release, following the protonation of the carbonate ion.

Amino Acid Substitution↗

PubMed2003

Interlobe communication in human serum transferrin: metal binding and conformational dynamics investigated by electrospray ionization mass spectrometry.

Human serum transferrin (hTF) is an iron transport protein, comprising two lobes (N and C), each containing a single metal-binding center. Despite substantial structural similarity between the two lobes, studies have demonstrated the existence of significant differences in their metal-binding properties. The nature of these differences has been elucidated through the use of electrospray ionization mass spectrometry to study both metal retention and conformational properties of hTF under a variety of conditions. In the absence of chelating agents or nonsynergistic anions, the diferric form of hTF remains intact until the pH is lowered to 4.5. The monoferric form of hTF retains the compact conformation until the pH is lowered to 4.0, whereas the apoprotein becomes partially unfolded at pH as high as 5.5. Selective (lobe-specific) modulation of the iron-binding properties of hTF using recombinant forms of the protein (in which the pH-sensitive elements in each lobe were mutated) verifies that the N-lobe of the protein has a lower affinity for ferric ion. Surprisingly, the apo-N-lobe is significantly less flexible compared to the apo-C-lobe. Furthermore, the conformation of the iron-free N-lobe is stabilized when the C-lobe contains iron, confirming the existence of an interlobe interaction within the protein. The experimental results provide strong support for the earlier suggestion that hTF interacts with its receptor (TFR) primarily through the C-lobe both at the cell surface and inside the endosome.

Acids↗

PubMed2003

Investigation of the mechanism of iron release from the C-lobe of human serum transferrin: mutational analysis of the role of a pH sensitive triad.

The transferrins (TFs) are a family of proteins that are widely distributed in vertebrates, where they serve a major role in iron binding and transport. Most TFs are composed of two homologous lobes, the N- and C-lobes, each able to bind a single iron atom. Human serum transferrin (hTF) binds iron in the blood and delivers it to actively dividing cells; through the process of receptor-mediated endocytosis, diferric hTF in the serum (pH approximately 7.4) binds to specific TF receptors on the cell surface and is internalized, whereupon a pH drop in the endosome (pH approximately 5.6) facilitates iron release. Many factors affect the rate of iron release, including pH, chelator, temperature, salt, and lobe-lobe interactions. We, and others, have actively studied the mechanism of iron release from the recombinant N-lobe of hTF; in contrast, the exact details of iron release from the C-lobe have remained less well characterized but appear to differ from those found for the N-lobe. Recently, to simplify the purification protocol, we have expressed and purified full-length recombinant hTF containing an N-terminal hexahistidine tag [Mason et al. (2002) Biochemistry 41, 9448-9454]. In the present work, we have expressed a full-length recombinant hTF containing a K206E mutation such that the N-lobe does not readily release iron. The resulting full-length hTF allows us to focus on the C-lobe and to study the effects of mutations introduced into the C-lobe. The success of this strategy is documented and in vitro mutagenesis is used to identify three residues in the C-lobe that are critical for iron-release. Although the importance of this triad is unequivocally demonstrated, further studies are needed to completely elucidate the mechanism of iron release from the C-lobe of hTF. In addition, the striking difference in the effect of increasing salt concentrations on iron release from the two lobes of hTF is further documented in the present work.

Amino Acid Substitution↗

PubMed2002

The position of arginine 124 controls the rate of iron release from the N-lobe of human serum transferrin. A structural study.

Human serum transferrin (hTF) is a bilobal iron-binding and transport protein that carries iron in the blood stream for delivery to cells by a pH-dependent mechanism. Two iron atoms are held tightly in two deep clefts by coordination to four amino acid residues in each cleft (two tyrosines, a histidine, and an aspartic acid) and two oxygen atoms from the "synergistic" carbonate anion. Other residues in the binding pocket, not directly coordinated to iron, also play critical roles in iron uptake and release through hydrogen bonding to the liganding residues. The original crystal structures of the iron-loaded N-lobe of hTF (pH 5.75 and 6.2) revealed that the synergistic carbonate is stabilized by interaction with Arg-124 and that both the arginine and the carbonate adopt two conformations (MacGillivray, R. T. A., Moore, S. A., Chen, J., Anderson, B. F., Baker, H., Luo, Y. G., Bewley, M., Smith, C. A., Murphy, M. E., Wang, Y., Mason, A. B., Woodworth, R. C., Brayer, G. D., and Baker, E. N. (1998) Biochemistry 37, 7919-7928). In the present study, we show that the two conformations are also found for a structure at pH 7.7, indicating that this finding was not strictly a function of pH. We also provide structures for two single point mutants (Y45E and L66W) designed to force Arg-124 to adopt each of the previously observed conformations. The structures of each mutant show that this goal was accomplished, and functional studies confirm the hypothesis that access to the synergistic anion dictates the rate of iron release. These studies highlight the importance of the arginine/carbonate movement in the mechanism of iron release in the N-lobe of hTF. Access to the carbonate via a water channel allows entry of protons and anions, enabling the attack on the iron.

Amino Acid Sequence↗

PubMed2002

Differential effect of a his tag at the N- and C-termini: functional studies with recombinant human serum transferrin.

Attachment of a cleavable hexa His tag is a common strategy for the production of recombinant proteins. Production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa His tag at the carboxyl terminus, has been described [Mason et al. (2001) Prot. Exp. Purif 23, 142-150]. More recently, hTF-NG with an amino-terminal His tag and a factor Xa cleavage site has been expressed (>30 mg/L) in baby hamster kidney cells and purified from the tissue culture medium. Although it is frequently assumed that addition of a His tag has little or no effect on function, this is not always confirmed experimentally. In the present study, in vitro quantitative data clearly shows that the presence of the C-terminal His tag has an effect on the release of iron from recombinant hTF at pH 7.4 and 5.6. Measurement of the rate of release from both the N- and C-lobes is reduced 2-4-fold. These findings provide further compelling evidence that the two lobes communicate with each other and highlight the importance of the C-terminal portion of the C-terminal lobe in this interaction. In contrast to these results, we demonstrate that the presence of a His tag at the N-terminus of hTF has no effect on the rate of iron release from either lobe. In a competition experiment, both unlabeled N- and C-terminal His-tagged constructs were equally effective at inhibiting the binding of radio-iodinated diferric glycosylated hTF from a commercial source to receptors on HeLa cells as the unlabeled recombinant diferric hTF-NG control. Thus, the presence of a His tag at either the N- or C-terminus of hTF-NG has no apparent effect on the ability of these hTF species to bind to transferrin receptors.

Base Sequence↗