Cobalt and the Iron Acquisition Pathway: Competition towards Interaction with Receptor 1

doi:10.1016/j.jmb.2008.05.045

Journal of Molecular Biology

Volume 380, Issue 5, 25 July 2008, Pages 900-916

https://doi.org/10.1016/j.jmb.2008.05.045 Get rights and content

Abstract

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO₃^2 − to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co^3 + and CO₃^2 − from CoNtaCO₃^2 − to the metal-binding site in the C-lobe of T: direct rate constant, k₁ = (1.1 ± 0.1) × 10⁶ M^− 1 s^− 1; reverse rate constant, k_− 1 = (1.9 ± 0.6) × 10⁶ M^− 1 s^− 1; and equilibrium constant, K = 1.7 ± 0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k₂ = (3 ± 0.3) × 10⁶ M^− 1 s^− 1; reverse rate constant, k_− 2 = (1.6 ± 0.3) × 10^− 2 s^− 1; and equilibrium constant, K_2a = 5.3 ± 1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo₂) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo₂ with the helical domain of R1: direct rate constant, k₃ = (4.4 ± 0.6) × 10¹⁰ M^− 1 s^− 1; reverse rate constant, k_− 3 = (3.6 ± 0.6) × 10⁴ s^− 1; and dissociation constant, K_1d = 0.82 ± 0.25 μM. The second is a very slow interaction of the N-lobe of TCo₂ with the protease-like domain of R1. This increases the stability of the protein–protein adduct by 30-fold with an average overall dissociation constant K_d = 25 ± 10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE—with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe—competes with iron-saturated transferrin in an unknown state towards interaction with R1.

Introduction

Transferrins and their receptors are the major iron transport system in vertebrates and invertebrates.¹ Human serum apotransferrin in an unknown state (T) is a glycoprotein that belongs to the transferrin superfamily.² It consists of a single amino-acid chain of about 700 residues organized in two similar, but not identical, lobes. Each lobe contains an iron-binding cleft, in which iron is coordinated to four protein ligands: the carboxylate of an aspartate, the imidazole of a histidine, and two phenolates of two tyrosines. Iron is also coordinated to a synergistic carbonate without which the protein loses its affinity for the metal.3, 4, 5 When transferrin is iron-loaded, it is recognized by complete homodimeric transferrin receptor 1 in an unknown state (R1), which is anchored in the plasma membrane.6, 7, 8, 9 We recently established a mechanism for iron uptake by transferrin¹⁰ and showed that both C-lobe iron-loaded transferrin and iron-saturated transferrin interact with R1.11, 12

R1 is a 190-kDa homodimeric protein that is arranged in two subunits linked by two disulfide bridges. Each of the subunits consists of a transmembrane and a cytoplasmic endodomain of about 11 kDa and a soluble ectodomain directed toward the biological fluid.⁷ R1 is arranged in four domains: the helical domain, the apical domain, the protease-like domain (which is close to the plasma membrane), and, finally, the endodomain.7, 9

T forms complexes with > 40 different metals.¹³ It was, therefore, considered to be involved in their incorporation into the organism through the iron acquisition pathway.13, 14, 15, 16 However, iron transport from the bloodstream to cytoplasm occurs in three apparent steps: (i) complex formation between serum transferrin and iron(III);¹⁰ (ii) interaction between iron-loaded T and receptor 1; and (iii) iron loss from the receptor on interaction with holotransferrin in mildly acidic media.6, 11, 12 Therefore, a metal can perturb the iron acquisition pathway or eventually be incorporated if it obeys at least two of these essential rules: (i) formation of a strong complex with T, and (ii) interaction of this complex with R1. Can this be the case with cobalt, and can the mechanisms of interaction of metal-loaded transferrins with R1 help us to understand the inhibition of the main iron acquisition pathway by other proteins such as HFE?17, 18

Cobalt is the essential trace element of vitamin B₁₂ (cobalamin), the deficiency of which results in pernicious anemia.¹⁹ Cobalt can be used in cancer chemotherapy²⁰ and, as its radioactive isotope, it is used in the diagnosis of hematopoietic disorders.²¹ This element can be toxic and accumulates in the liver with a half-life of about 10 years.²² Cobalt is known to form complexes with T,14, 23, 24 with an affinity constant estimated to be close to that of iron (∼ 10²¹ M).¹³

In a series of recent articles, we established the mechanisms of complex formation between serum transferrin and a series of metallic elements such as Fe(III), Al(III), Bi(III), and Ga(III).10, 25, 26, 27 We showed that although the mechanisms of complex formation between serum T and these different metals are not identical, these metal uptakes always take place sequentially, with a first complex being formed between the protein ligands in the C-lobe iron cleft on interaction with bicarbonate. This process usually takes place in the range of 0.1 s to several seconds. It induces a series of changes in the conformation of the protein that affect the N-lobe, thus favoring a second metal uptake.10, 25, 26, 27 We also showed that bismuth and gallium-loaded transferrins interact with R1, whereas aluminum-loaded T does not interact with R1.25, 26, 27 Besides aluminum, the overall affinity of R1 for these metal-loaded transferrins is always at least 3 orders of magnitude lower than that of iron-saturated transferrin in an unknown state (TFe₂). Moreover, only the C-lobe of these metal-loaded transferrins seems to interact with R1. We presumed that this interaction can be sufficient to permit the incorporation of bismuth and gallium by the receptor-mediated iron acquisition pathway.26, 27

In this article, we expand our investigations to cobalt and use the techniques and methods of chemical relaxation28, 29 to determine the mechanism of cobalt uptake by T and to investigate the interaction of the cobalt-loaded T with R1. A generalization of this mechanism to the interaction of other metal-loaded transferrins or altogether different proteins (such as HFE) with R1 and to the influence of such interactions on iron transport is attempted.

Section snippets

Results

The cobalt donor to T used in our experiment is the mixed carbonate nitrilotriacetate sodium complex (CoNtaCO₃^2 −).30, 31 This complex was used because of its well-identified structure when compared to other undefined cobalt(III) complexes, such as that of nitrilotriacetate (Nta^3 −).³⁰ The transferrin receptor used is the noncleaved complete homodimer either in its native form or rhodamine-labeled.¹¹ Spectrophotometric detection was used in all thermodynamic and kinetic runs. Indeed, T,

Cobalt(III) Uptake by Transferrin

Adding CoNtaCO₃^2 − complex to a solution of apotransferrin leads to important modifications in both the absorption and the fluorescence emission spectra of the protein (Fig. 1). These are manifested by a decrease in the fluorescence emission, accompanied by a bathochromic shift of 7 nm and by an already reported modification in the absorption spectra with a typical band at 405 nm.14, 32 Moreover, when cobalt-saturated T,¹⁴ a transferrin that reacted with a single cobalt equivalent¹⁰

Interaction of Cobalt(III)-Loaded Transferrin with Receptor 1

At a fixed pH value, adding TCo₂ to R1 in the presence of 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS) leads to a decrease in fluorescence intensity accompanied by a red shift in the maximum of emission. Similar spectral modifications were also observed when iron-loaded transferrin (TFe₂) was added to R1.¹¹ As in this last case, we ascribe this fluorescence variation to the molecular interaction of the receptor with TCo₂ (Eq. (24)): $R 1 + n {TCo}_{2} ⇌ (R 1) - ({TCo}_{2})_{n}$ in which (R1) −

Discussion

Iron uptake by T requires a synergistic anion.³⁶ Carbonate is a natural synergistic anion and is also required for the uptake of metals other than iron by apotransferrin.25, 26, 27 In the absence of carbonate and with Nta^3 −, a mixed FeNta–transferrin complex is formed.³⁷ In this ternary mixed complex, Nta^3 − is rapidly and easily replaced by carbonate.³⁵ In the presence of the synergistic bicarbonate, with iron, bismuth, and gallium, the first step in metal uptake by T is always a very fast

Conclusion

Cobalt uptake by transferrin occurs by mechanisms different from the uptake of other metals such as iron, gallium, bismuth, and aluminum. Both the C-lobe and the N-lobe of the cobalt-loaded transferrin interact with receptor 1 by a mechanism close to that of holotransferrin with, however, a similar affinity for the C-lobe and a lower affinity for the N-lobe. Nonetheless, in iron delivery by the transferrin R1-mediated iron acquisition pathway, the essential step is that of the interaction of

Materials and Methods

Better than 98% pure human serum apotransferrin (Sigma) was further purified by published procedures; its purity was checked spectrophotometrically and by urea polyacrylamide gel electrophoresis.10, 33 KCl (Merck Suprapur); NaOH and HCl (Merck Titrisol); ethylenediaminetetraacetic acid (Merck Titriplex); FeCl₃, CoCl₂, trisodium citrate, acetic acid (96%), and sodium acetate (Merck); sodium bicarbonate and 35% H₂O₂ (Aldrich); glycerol, urea (electrophoresis reagent), SDS, boric acid, CHAPS

Acknowledgements

The authors are grateful to Dr. John S. Lomas for constructive discussions, to the Jean Rostand Maternity Hospital for their gift of placentas, and to Novartis for their gift of desferrioxamine.

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In this study, we aimed to expand our knowledge of this phenomenon by designing a computational method to identify genes that may be related to both HCV and trace element metabolic processes. The searching procedure included three stages. First, a shortest path algorithm was applied to a large network, constructed by protein-protein interactions, to identify potential genes of interest. Second, a permutation test was executed to exclude false discoveries. Finally, some rules based on the betweenness and associations between candidate genes and HCV and trace elements were built to select core genes among the remaining genes.
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Human serum transferrin (hTf) is a single-chain bilobal glycoprotein that efficiently delivers iron to mammalian cells by endocytosis via the transferrin/transferrin receptor system. While extensive studies have been directed towards the study of ferric ion binding to hTf, ferrous ion interactions with the protein have never been firmly investigated owing to the rapid oxidation of Fe^II to Fe^III and the difficulty in maintaining a fully anaerobic environment. Here, the binding of Fe^2 + and Zn^2 + ions to hTf has been studied under anaerobic and aerobic conditions, respectively, in the presence and absence of bicarbonate by means of isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The ITC data indicate the presence of one class of strong binding sites with dissociation constants of 25.2 nM for Fe^2 + and 6.7 nM for Zn^2 + and maximum binding stoichiometries of 1 Zn^2 + (or 1 Fe^2 +) per hTf molecule. With either metal, the binding interaction was achieved by both favorable enthalpy and entropy changes (ΔH⁰ ~ − 12 kJ/mol and ΔS⁰ ~ 106 J/mol·K for Fe^2 + and ΔH⁰ ~ − 18 kJ/mol and ΔS⁰ ~ 97 J/mol·K for Zn^2 +). The large and positive entropy values are most likely due to the change in the hydration of the protein and the metal ions upon interaction. Rapid kinetics stopped-flow fluorescence spectroscopy revealed two different complexation mechanisms with different degrees of conformational changes upon metal ion binding. Our results are discussed in terms of a plausible scenario for iron dissociation from transferrin by which the highly stable Fe^3 +–hTf complex might be reduced to the more labile Fe^2 + ion before iron is released to the cytosol.
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Journal of Molecular Biology

Cobalt and the Iron Acquisition Pathway: Competition towards Interaction with Receptor 1

Abstract

Introduction

Section snippets

Results

Cobalt(III) Uptake by Transferrin

Interaction of Cobalt(III)-Loaded Transferrin with Receptor 1

Discussion

Conclusion

Materials and Methods

Acknowledgements

J. Biol. Chem.

Cell

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Exp. Hematol.

Chem. Biol. Interact.

J. Biol. Chem.

Polyhedron

Bioorg. Med. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Inorganic Biochemistry of Iron Metabolism

Structure of Haemophilus influenzae Fe(+3)-binding protein reveals convergent evolution within a superfamily

Nat. Struct. Biol.

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

J. Biol. Chem.

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

Biochemistry

pH and the recycling of transferrin during receptor-mediated endocytosis

Proc. Natl Acad. Sci. USA

Crystal structure of the ectodomain of human transferrin receptor

Science

Transferrin's mechanism of interaction with receptor 1

Biochemistry

Transferrin as a metal ion mediator

Chem. Rev.