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

Abstract

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.

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

“…A second, functionally diverse class of carboxylate-bridged diiron proteins (2-4, 65) features a helical Glu-Xxx-Xxx-His (EXXH) motif, with two rather than three residues between the liganding side chains. We have conducted a retrostructural analysis (27) of six members of this class, including three ferroxidases [ferritin (66), bacterioferritin (67), and rubrerythrin (68, 69)], ribonucleotide reductase R2 subunit (R2) (70), ⌬ 9 ACP desaturase (71), and the catalytic subunit of methane monooxygenase (72). Although there is less than 5% sequence identity common to all members of this class, their active sites are housed within a very simple pseudo-222-symmetric four-helix bundle.…”

The crystal structure of TGF‐β3 and comparison to TGF‐β2: Implications for receptor binding

“…These observations indicate that the trigonal TGF-83 crystals are rather stabilized by the attraction of complementary charged surfaces and hydrophobic interactions than by the effects of individual residues. In the trigonal crystal form, dioxane might be as important for keeping symmetry-related molecules together by hydrophobic interactions as, for example, Ca2+ ions are for the formation of ferritin crystals (Lawson et al, 1991) or phosphate ions are for bovine pancreatic trypsin inhibitor crystals (Wlodawer, 1984).…”

Direct observation of the iron binding sites in a ferritin

“…The ability of the protein to catalyse Fe(I1) oxidation is well established [4,5], but the catalytic mechanism and the subsequent stages leading to mineralisation are not clearly defined. The use of site-directed mutagenesis has implicated a number of residues in iron-binding [4-71, but one of the reasons for the lack of direct information on ironbinding positions is that crystallisation of mammalian ferritins has always involved metal ions, usually Cd2+ or Ca2' [1,8]. Such metals compete with binding of Fe2' or Fe3' ions and thus prevent the unequivocal assignment of their sites.…”