Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution.

Mammalian ferritins are 24-mers assembled from two types of polypeptide chain which provide the molecule with different functions. H(eavy) chains catalyse the first step in iron storage, the oxidation of iron(II). L(ight) chains promote the nucleation of the mineral ferrihydrite enabling storage of iron(III) inside the protein shell. We report here the comparison of the three-dimensional structures of recombinant human H chain (HuHF) and horse L chain (HoLF) ferritin homopolymers, which have been refined at 1.9 A resolution. There is 53% sequence identity between these molecules, and the two structures are very similar, the H and L subunit alpha-carbons superposing to within 0.5 A rms deviation with 41 water molecules in common. Nevertheless, there are significant important differences which can be related to differences in function. In particular, the centres of the four-helix bundles contain distinctive groups of hydrophilic residues which have been associated with ferroxidase activity in H chains and enhanced stability in L chains. L chains contain a group of glutamates associated with mineralisation within the iron storage cavity of the protein.

Bovine beta-lactoglobulin at 1.8 A resolution--still an enigmatic lipocalin.

BACKGROUND: beta-Lactoglobulin (beta-Lg) is the major whey protein in the milk of ruminants and many other mammals. Its function is not known, but it undergoes at least two pH-dependent conformational changes which may be important. Bovine beta-Lg crystallizes in several different lattices, and medium-resolution structures of orthorhombic lattice Y and trigonal lattice Z have been published. Triclinic lattice X and lattice Z crystals grow at pH values either side of the pH at which one of the pH-induced conformational changes occurs. A full understanding of the structure is needed to help explain both the conformational changes and the different denaturation behaviour of the genetic variants. RESULTS: We have redetermined the structure of beta-Lg lattice Z at 3.0 A resolution by multiple isomorphous replacement and have partially refined it (R factor = 24.8%). Using the dimer from this lattice Z structure as a search model, the triclinic crystal form grown at pH 6.5 (lattice X) has been solved by molecular replacement. Refinement of lattice X at 1.8 A resolution gave an R factor of 18.1%. The structure we have determined differs from previously published structures in several ways. CONCLUSIONS: Incorrect threading of the sequence in the published structures of beta-Lg affects four of the nine beta strands. The basic lipocalin fold of the polypeptide chain is unchanged, however. The relative orientation of the monomers in the beta-Lg dimer differs in the two lattices. On raising the pH, there is a rotation of approximately 5 degrees, which breaks a number of intersubunit hydrogen bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the beta strand with the free thiol, each of which shows genetic variation.

Iron (II) oxidation and early intermediates of iron-core formation in recombinant human H-chain ferritin.

The paper describes a study of Fe(II) oxidation and the formation of Fe(III)-apoferritin complexes in recombinant human H-chain ferritin and its variants. The effects of site-directed changes in the conserved residues associated with a proposed ferroxidase centre have been investigated. A change in any of these residues is shown to reduce the rate of Fe(II) oxidation, confirming the importance of the ferroxidase centre in the catalysis of Fe(II) oxidation. Mössbauer and u.v.-difference spectroscopy show that in the wild-type protein Fe(II) oxidation gives rise to Fe(III) monomers, dimers and larger clusters. The formation of Fe(III) mu-oxo-bridged dimers occurs at the ferroxidase centre and is associated with fast oxidation: in three variants in which Fe(II) oxidation is especially slow, no Fe(III) dimers are seen. Within the time scale 0.5-20 min in wild-type human H-chain ferritin, dimer formation precedes that of the monomer and the progression dimer-->monomer-->cluster is observed, although not to completion. In a preliminary investigation of oxidation intermediates using a stopped-flow instrument, an Fe(III)-tyrosine complex reported by Waldo et al. (1993), is attributed to Tyr-34, a residue at the ferroxidase centre. The Fe(III)-Tyr-34 complex, forms in 0.5 s and then decays, as dimer absorbance increases. The relationship between Fe(III)-tyrosinate and the formation of Fe(III) dimers is uncertain.

Defining the roles of the threefold channels in iron uptake, iron oxidation and iron-core formation in ferritin: a study aided by site-directed mutagenesis.

This paper aims to define the role of the threefold intersubunit channels in iron uptake and sequestration processes in the iron-storage protein, ferritin. Iron uptake, measured as loss of availability of Fe(II) to ferrozine (due to oxidation), has been studied in recombinant human H-chain ferritins bearing amino acid substitutions in the threefold channels or ferroxidase centres. Similar measurements with recombinant horse L-chain ferritin are compared. It is concluded that significant Fe(II) oxidation occurs only at the H-chain ferroxidase centres and not in the threefold channels, although this route is used by Fe(II) for entry. Investigations by Mössbauer and u.v.-difference spectroscopy show that part of the iron oxidized by H-chain ferritin returns to the threefold channels as Fe(III). This monomeric Fe(III) can be displaced by addition of Tb(III). Fe(III) also moves into the cavity for formation of the iron-core mineral, ferrihydrite. Iron incorporated into ferrihydrite becomes kinetically inert.

Haem and non-haem iron sites in Escherichia coli bacterioferritin: spectroscopic and model building studies.

The bacterioferritin (BFR) of Escherichia coli is an iron-storage protein containing 24 identical subunits and between three and 11 protohaem IX groups per molecule. Titration with additional haem gave a maximum loading of 12-14 haems per molecule. The e.p.r. spectra and magnetic c.d. spectra of the protein-bound haem show it to be low-spin Fe(III), and coordinated by two methionine residues as previously reported for BFRs isolated from Pseudomonas aeruginosa and Azotobacter vinelandii [Cheesman, Thomson, Greenwood, Moore and Kadir, Nature (London) (1990) 346, 771-773]. A recent sequence alignment indicated that BFR may be structurally related to ferritin. The molecular model proposed for E. coli BFR has a four-alpha-helix-bundle subunit conformation and a quaternary structure similar to those of mammalian ferritins. In this model there are two types of hydrophobic pocket within which two methionine residues are correctly disposed to bind haem. The e.p.r. spectra also reveal a monomeric non-haem Fe(III) species with spin, S = 5/2. On the basis of sequence comparisons, a ferroxidase centre has recently been proposed to be present in BFR [Andrews, Smith, Yewdall, Guest and Harrison (1991) FEBS Lett. 293, 164-168] and the possibility that this Fe(III) ion may reside at or near the ferroxidase centre is discussed.

Evidence of H- and L-chains have co-operative roles in the iron-uptake mechanism of human ferritin.

The ability to incorporate iron in vitro was studied in homopolymers of human ferritin L-chain, human ferritin H-chain and its variants and in homopolymer mixtures. The H-chain variants carried amino acid substitutions in the ferroxidase centre and/or in carboxy residues on the cavity surface. Iron incorporation was examined by gel electrophoresis of the reaction products by staining for iron and protein. It was found that inactivation of the ferroxidase centre combined with the substitution of four carboxy groups on the cavity abolished the ability of H-chain ferritin to incorporate iron. Competition experiments with limited amounts of iron showed that, at neutral pH, L-chain ferritin is more efficient in forming iron cores than the H-chain variants altered at the ferroxidase activity or in the cavity. Competition experiments at pH 5.5 demonstrated that L-chain apoferritin is able to incorporate iron only when in the presence of H-chain variants with ferroxidase activity. The results indicate that L-chain apoferritin has a higher capacity than the H-chain apoferritin to induce iron-core nucleation, whereas H-chain ferritin is superior in promoting Fe(II) oxidation. The finding of cooperative roles of the H- and L-chains in ferritin iron uptake provides a clue to understanding the biological function of isoferritins.

Amino-acid sequence and predicted three-dimensional structure of pea seed (Pisum sativum) ferritin.

The iron storage protein, ferritin, is widely distributed in the living kingdom. Here the complete cDNA and derived amino-acid sequence of pea seed ferritin are described, together with its predicted secondary structure, namely a four-helix-bundle fold similar to those of mammalian ferritins, with a fifth short helix at the C-terminus. An N-terminal extension of 71 residues contains a transit peptide (first 47 residues) responsible for plastid targetting as in other plant ferritins, and this is cleaved before assembly. The second part of the extension (24 residues) belongs to the mature subunit; it is cleaved during germination. The amino-acid sequence of pea seed ferritin is aligned with those of other ferritins (49% amino-acid identity with H-chains and 40% with L-chains of human liver ferritin in the aligned region). A three-dimensional model has been constructed by fitting the aligned sequence to the coordinates of human H-chains, with appropriate modifications. A folded conformation with an 11-residue helix is predicted for the N-terminal extension. As in mammalian ferritins, 24 subunits assemble into a hollow shell. In pea seed ferritin, its N-terminal extension is exposed on the outside surface of the shell. Within each pea subunit is a ferroxidase centre resembling those of human ferritin H-chains except for a replacement of Glu-62 by His. The channel at the 4-fold-symmetry axes defined by E-helices, is predicted to be hydrophilic in plant ferritins, whereas it is hydrophobic in mammalian ferritins.

Spectroscopic identification of the haem axial ligands of haemoferritin and location of possible haem-binding sites in ferritin by molecular modelling.

Horse spleen ferritin will bind up to 16 protoporphyrin IX haem groups per 24 subunits in vitro [Kadir & Moore (1990) FEBS Lett. 276, 81-84] at a site that causes the haem to be low spin for both ferric and ferrous states. E.p.r. spectra at 10 K of the oxidized form of the resulting haemoferritin gives g values of 2.93, 2.26 and 1.55, characteristic of low-spin haem. The near-i.r. magnetic circular dichroism spectrum shows a porphyrin-to-ferric charge-transfer band at 1590 nm. The spectroscopic parameters indicate that the haem group is probably bound by two histidine ligands. Molecular modelling studies reveal one type of potential haem-binding site in horse L-chain ferritin with bis-histidine co-ordination. This is an intersubunit site which lies in a pocket within the ferritin protein shell in the region of the 3-fold channel. The ligands are His-114 and His-124 in horse L-chain. A second possible set of sites in human H-chain ferritin involves His-60 residues in the pockets between pairs of subunits. These are considered less likely sites of haem occupancy. There are three of the intersubunit sites in horse L-chain ferritin at each of the eight 3-fold channels. We propose that conformational crowding between haem-binding sites at a given channel prevents more than two haems per channel being bound.

Structure, function, and evolution of ferritins.

The ferritins of animals and plants and the bacterioferritins (BFRs) have a common iron-storage function in spite of differences in cytological location and biosynthetic regulation. The plant ferritins and BFRs are more similar to the H chains of mammals than to mammalian L chains, with respect to primary structure and conservation of ferroxidase center residues. Hence they probably arose from a common H-type ancestor. The recent discovery in E. coli of a second type of iron-storage protein (FTN) resembling ferritin H chains raises the question of what the relative roles of these two proteins are in this organism. Mammalian L ferritins lack ferroxidase centers and form a distinct group. Comparison of the three-dimensional structures of mammalian and invertebrate ferritins, as well as computer modeling of plant ferritins and of BFR, indicate a well conserved molecular framework. The characterisation of numerous ferritin homopolymer variants has allowed the identification of some of the residues involved in iron uptake and an investigation of some of the functional differences between mammalian H and L chains.

Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins.

Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains. The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown. Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions. In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains. In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin. The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain. One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold.

Mechanism of catalysis of Fe(II) oxidation by ferritin H chains.

Recombinant H chain ferritins bearing site-directed amino acid substitutions at their ferroxidase centres have been used to study the mechanism of catalysis of Fe(II) oxidation by this protein. UV-difference spectra have been obtained at various times after the aerobic addition of Fe(II) to the recombinants. These indicate that the first product of Fe(II) oxidation by wild type H chain apoferritin is an Fe(III) mu-oxo-bridged dimer. This suggests that fast oxidation is achieved by 2-electron transfer from two Fe(II) to dioxygen. Modelling of Fe(III) dimer binding to human H chain apoferritin shows a solvent-accessible site, which resembles that of ribonucleotide reductase in its ligands. Substitution of these ligands by other amino acids usually prevents dimer formation and leads to greatly reduced Fe(II) oxidation rates.

Bacterioferritins and ferritins are distantly related in evolution. Conservation of ferroxidase-centre residues.

Iron-storage proteins can be divided into two classes; the bacterioferritins and ferritins. In spite of many apparent structural and functional analogies, no significant amino acid sequence similarity has been detected previously. This report now reveals a distant evolutionary relationship between bacterioferritins and ferritins derived by 'Profile Analysis'. Optimum alignment of bacterioferritin and ferritin sequences suggests that key residues of the ferroxidase centres of ferritins are conserved in bacterioferritins.

Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts.

Ferritin is important in iron homeostasis. Its twenty-four chains of two types, H and L, assemble as a hollow shell providing an iron-storage cavity. Ferritin molecules in cells containing high levels of iron tend to be rich in L chains, and may have a long-term storage function, whereas H-rich ferritins are more active in iron metabolism. The molecular basis for the greater activity of H-rich ferritins has until now been obscure, largely because the structure of H-chain ferritin has remained unknown owing to the difficulties in obtaining crystals ordered enough for X-ray crystallographic analysis. Here we report the three-dimensional structure of a human ferritin H-chain homopolymer. By genetically engineering a change in the sequence of the intermolecular contact region, we obtained crystals isomorphous with the homologous rat L ferritin and of high enough quality for X-ray diffraction analysis. The X-ray structure of human H ferritin shows a novel metal site embedded within each of its four-helix bundles and we suggest that ferroxidase activity associated with this site accounts for its rapid uptake of iron.

Identification of the ferroxidase centre in ferritin.

Ferroxidase activity in human H-chain ferritin has been studied with the aid of site-directed mutagenesis. A site discovered by X-ray crystallography has now been identified as the ferroxidase centre. This centre is present only in H-chains and is located within the four-helix bundle of the chain fold.