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S J Yewdall

Publications and source records attributed to S J Yewdall.

9 recordsLinked to original sources
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Apoferritins↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Alanine↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

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.

Amino Acid Sequence↗
PubMedDate not supplied

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.

Amino Acid Sequence↗