Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala

Béatrice Langlois D'Estaintot, Paolo Santambrogio, Thierry Granier, Bernard Gallois, Jean Marc Chevalier, Gilles Précigoux, Sonia Levi, Paolo Arosio

Research output: Contribution to journalArticle

Abstract

Mitochondrial ferritin is a recently identified protein precursor encoded by an intronless gene. It is specifically taken up by the mitochondria and processed to a mature protein that assembles into functional ferritin shells. The full mature recombinant protein and its S144A mutant were produced to study structural and functional properties. They yielded high quality crystals from Mg(II) solutions which diffracted up to 1.38Å resolution. The 3D structures of the two proteins resulted very similar to that of human H-ferritin, to which they have high level of sequence identity (∼80%). Metal-binding sites were identified in the native crystals and in those soaked in Mn(II) and Zn(II) solutions. The ferroxidase center binds binuclear iron at the sites A and B, and the structures showed that the A site was always fully occupied by Mg(II), Mn(II) or Zn(II), while the occupancy of the B site was variable. In addition, distinct Mg(II) and Zn(II)-binding sites were found in the 3-fold axes to block the hydrophilic channels. Other metal-binding sites, never observed before in H-ferritin, were found on the cavity surface near the ferroxidase center and near the 4-fold axes. Mitochondrial ferritin showed biochemical properties remarkably similar to those of human H-ferritin, except for the difficulty in renaturing to yield ferritin shells and for a reduced (∼41%) rate in ferroxidase activity. This was partially rescued by the substitution of the bulkier Ser144 with Ala, which occurs in H-ferritin. The residue is exposed on a channel that connects the ferroxidase center with the cavity. The finding that the mutation increased both catalytic activity and the occupancy of the B site demonstrated that the channel is functionally important. In conclusion, the present data define the structure of human mitochondrial ferritin and provide new data on the iron pathways within the H-type ferritin shell.

Original languageEnglish
Pages (from-to)277-293
Number of pages17
JournalJournal of Molecular Biology
Volume340
Issue number2
DOIs
Publication statusPublished - Jul 2 2004

Fingerprint

Apoferritins
Ceruloplasmin
Ferritins
Binding Sites
Iron
Metals
Protein Precursors
Recombinant Proteins
Mitochondria
Proteins
human mitochondrial ferritin
Mutation
Genes

Keywords

  • crystal structure
  • ferritin
  • ferroxidase activity
  • metal-binding sites
  • mitochondria
  • MtF, mitochondrial ferritin
  • rHuHF, recombinant human H-ferritin
  • rMtF, recombinant MtF
  • ROS, reactive oxygen species

ASJC Scopus subject areas

  • Virology

Cite this

Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala. / D'Estaintot, Béatrice Langlois; Santambrogio, Paolo; Granier, Thierry; Gallois, Bernard; Chevalier, Jean Marc; Précigoux, Gilles; Levi, Sonia; Arosio, Paolo.

In: Journal of Molecular Biology, Vol. 340, No. 2, 02.07.2004, p. 277-293.

Research output: Contribution to journalArticle

D'Estaintot, Béatrice Langlois ; Santambrogio, Paolo ; Granier, Thierry ; Gallois, Bernard ; Chevalier, Jean Marc ; Précigoux, Gilles ; Levi, Sonia ; Arosio, Paolo. / Crystal structure and biochemical properties of the human mitochondrial ferritin and its mutant Ser144Ala. In: Journal of Molecular Biology. 2004 ; Vol. 340, No. 2. pp. 277-293.
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abstract = "Mitochondrial ferritin is a recently identified protein precursor encoded by an intronless gene. It is specifically taken up by the mitochondria and processed to a mature protein that assembles into functional ferritin shells. The full mature recombinant protein and its S144A mutant were produced to study structural and functional properties. They yielded high quality crystals from Mg(II) solutions which diffracted up to 1.38{\AA} resolution. The 3D structures of the two proteins resulted very similar to that of human H-ferritin, to which they have high level of sequence identity (∼80{\%}). Metal-binding sites were identified in the native crystals and in those soaked in Mn(II) and Zn(II) solutions. The ferroxidase center binds binuclear iron at the sites A and B, and the structures showed that the A site was always fully occupied by Mg(II), Mn(II) or Zn(II), while the occupancy of the B site was variable. In addition, distinct Mg(II) and Zn(II)-binding sites were found in the 3-fold axes to block the hydrophilic channels. Other metal-binding sites, never observed before in H-ferritin, were found on the cavity surface near the ferroxidase center and near the 4-fold axes. Mitochondrial ferritin showed biochemical properties remarkably similar to those of human H-ferritin, except for the difficulty in renaturing to yield ferritin shells and for a reduced (∼41{\%}) rate in ferroxidase activity. This was partially rescued by the substitution of the bulkier Ser144 with Ala, which occurs in H-ferritin. The residue is exposed on a channel that connects the ferroxidase center with the cavity. The finding that the mutation increased both catalytic activity and the occupancy of the B site demonstrated that the channel is functionally important. In conclusion, the present data define the structure of human mitochondrial ferritin and provide new data on the iron pathways within the H-type ferritin shell.",
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