Localized Unfolding at the Junction of Three Ferritin Subunits

How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >1011-fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a “shutter” that cytoplasmic factors might open or close to regulate iron release in vivo.

the rate of exit of iron from ferritin. When conserved leucine 134 was replaced by proline (L134P), the protein assembled, oxidized Fe(II), and mineralized Fe(III), but the time for complete dissolution of mineral (480 iron) in vitro was greatly decreased (5 min compared with 150 min for the parent protein). X-ray diffraction studies of crystals of H-L134P ferritin showed a flexible region localized near the termini of two subunit helices (C, D), which form the interfaces of subunit trimers and a channel. The results indicate that iron can exit from ferritin at the trimer subunit junction. A possible mechanism for regulated iron release in vivo could be localized disorder in the assembled protein, enhanced by cytoplasmic changes with effects analogous to the effect of H-L134P.
Kinetic Studies of Iron Uptake and Release-The method for iron uptake was described previously (13). In iron release experiments, apoferritins (2.08 M) were mineralized by the addition of a solution of ferrous sulfate at the iron/protein ϭ 480 in 0.1 M MOPS 1 (pH 7.0) and 0.2 M NaCl, followed by incubation for 2 h at room temperature and then incubation overnight at 4°C (11)(12)(13). Iron release was initiated by the addition of 2.5 mM bipyridyl, 2.5 mM FMN, and 2.5 mM NADH to reconstituted ferritin in 0.1 M MOPS (pH 7.0) and 0.2 M NaCl (14,15). The amount of iron released from ferritin was monitored at room temperature by the absorbance at 522 nm of the Fe(II)-bipyridyl complex.
X-ray Diffraction-Crystals of H-L134P, K82Q or H-L134P, R86Q ferritin were obtained by the hanging drop method. The crystallization conditions were optimized, beginning with the sparse-matrix sampling method (16), with the best H-L134P, K82Q crystals being obtained by mixing 5 l of a 10 mg/ml protein solution with an equal volume of 15% 2-methyl-2,4-pentanediol, 0.02 M CaCl 2 , 0.1 M sodium acetate buffer, pH 4.6. The best H-L134P, R86Q crystals were obtained in 25% isopropyl alcohol, 0.1 M sodium cacodylate, 0.2 M MgCl 2 . 2 Bipyramid-shaped crystals, ϳ0.5 ϫ 0.2 mm, formed within 2 weeks. Diffraction data were collected both with a conventional rotating anode x-ray generator and a Siemens area detector and on beamline X-12C at National Synchrotron Light Source, Brookhaven National Laboratory. The data were processed by XENGEN and DENZO, and refinements were carried out with X-PLOR (17). Data statistics and final refinement statistics are listed in Table I. Program O (18) was used for model building and fitting to the electron density map.

RESULTS
Ferritin subunits fold and assemble spontaneously as ironfree proteins (1, 2, 19 -24) with buffer in the cavity (22,23). 3 In nature, the iron minerals in ferritin range in average size from * This research was supported by National Institutes of Health Grants DK20251 (to H. T. and E. C. T.) and DK50727 (to D. S., Y. H., and N. M. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates ( 800 to 2500 iron/molecule and from microcrystalline to amorphous, particularly when the phosphate content is high (1, 2). The heterodispersity of mineral size in natural ferritin varies from ϳ10 to 4000 iron/molecule and is much greater than in minerals reconstituted to 480 iron/molecule from the iron-free protein (15). To study iron release, Fe(II) was added to the recombinant ferritin proteins to form iron minerals of constant average size (480 iron/molecule).
The role of ferritin in iron release was compared for proteins that differed over 1000-fold in iron uptake rates and in the mechanism of iron oxidation (fast-ferroxidase sites and nucleation sites present, slow-only nucleation sites present). The initial rates of iron oxidation by proteins with ferroxidase sites were 0.99 Ϯ 0.02 (A 650 /s) for H ferritin, 0.081 Ϯ 0.038 (A 550 /s) for H-L134P ferritin (13), and 1.55 Ϯ 0.22 (A 650 /s) for M ferritin (13). The slower rate of iron uptake/ferroxidation for the H-L134P protein is coupled to a shift in the absorbance maximum of the initial Fe(III) complex from 650 to 550 nm (Fig. 1) and a slower decay (12, 13), but the formation rate is still within the range for fast ferritins and 100-fold faster than L ferritin (13). L ferritin has no specific ferroxidation site, and the initial rate of oxidation is 0.0037 Ϯ 0.0017 (A 350 /s) (11).
Iron exit from the mineralized recombinant ferritins was triggered by reduction of Fe(III) with FMNH 2 /NADH and trapping the Fe(II) as the Fe(II)-bipyridyl complex. No Fe(II)-bipyridyl complex was detected until the reductant was added, and essentially all (96%) of the Fe(II)-bipyridyl complex that formed could be separated from the protein by ultrafiltration (data not shown). Previous studies have shown that the reductive release of iron is independent of reductant/chelator size (8,9). Rates of iron release from recombinant ferritins were biphasic ( Fig. 2A). There was little difference in iron release rates among the recombinant ferritins with natural sequences.
In contrast to the wild type proteins (H, M, L), substitution of leucine 134, conserved in all ferritins (1,2), with proline had a faster initial rate of iron release (Fig. 2) that was essentially monophasic. Complete dissolution of a ferritin mineral of 480 iron and release as Fe(II)-bipyridyl only required 5 min in the H-L134P protein compared with 150 min for the parent protein with L134.
Crystals formed by frog H ferritin with L134P showed localized changes in the subunit packing at the junction of three   (21)(22)(23)(24). 1.8-Å resolution synchrotron data collected at Brookhaven National Laboratory showed the same weak density region, and as expected for a truly disordered region, the better the data quality, the weaker the density in this region. The data from this region were excluded from the refinement.

DISCUSSION
The sites in the ferritin structure for reductive iron release are different from the rapid oxidation sites: iron release rates differed little in fast (H, M) and slow (L) ferritins with natural sequences (Fig. 2), but oxidation rates varied over a wide range (11)(12)(13). Rapid oxidation occurs in the center of the 4-helix bundle that forms the ferritin subunit and involves residues from helices A, B, and C (1,2,21,24). In contrast residue 134, which when changed to proline altered rates of iron exit, is near the N terminus of helix D (21)(22)(23)(24). The backbone nitrogen of Leu-134 is hydrogen-bonded to the carbonyl oxygen of Leu-129, whereas the side chain of Leu-134 has a hydrophobic interaction with Leu-110 in helix C. A kink in the backbone of the helix D adjacent to residue 134, produced by deviations from the standard dihedral angles at positions 132 and 133 (24), determines precise positioning of the interhelical and intersubunit interactions of the D helix near the subunit trimer interface. The L134P protein will have changed an intrasubunit hydrophobic interaction, hydrogen bonds, and intersubunit interactions.
H-L134P ferritin assembled from 24 subunits will have eight regions of local flexibility distributed symmetrically around the surface of the molecule caused by the changes at each junction of three subunits (Fig. 3). In other proteins, introduction of proline into a peptide can be without functional effect (26), the proteins adjusting conformation to accommodate the change (26 -28). However, in ferritin, the effect of the proline substitution is amplified by disrupting both cooperative interhelical and intersubunit interactions (Fig. 3). The unfolded structure at the 3-fold axes can alter the behavior of assembled ferritin H-L134P in solution. For example, the flexibility of the structure will propagate to the channels for iron entry; the mutation led to a shift in the absorbance maximum of the initial Fe(III) complex (Fig. 1), a decreased rate of oxidation (13), and a decreased decay rate of the initial Fe(III) complex that fortuitously permitted its identification as Fe(III)-tyrosine (12). Whether Fe(III)-tyrosine is specific to H-L134P ferritin protein or simply more readily detected in H-L134P protein because of the slower decay is not yet clear (see Ref. 3). 5 Disorder at the 3-fold axis of the assembled H-L134P protein appeared to increase the accessibility of the mineral core to solutes such as reductants and chelators (Fig. 2). The structure of ferritin H-L134P contrasts with the high degree of order in the same region of crystals of recombinant H or L ferritins from frog, horse, and human wild type (Fig. 4) (21)(22)(23)(24) or with amino acid substitutions in the A or B helices (22,23). Conserved residues with carboxylate side chains contributed from three subunits line the channel at the 3-fold axis (1,3,(21)(22)(23)(24)34), which is the site for both iron entry (1, 2, 29 -33, 35) and iron exit (Figs. 2-4). In addition to metal ions, the carboxylate side chains at the junction of three subunits could facilitate exit and entry of protons involved in mineralization (hydrolysis) and mineral dissolution (ϳ2.5 H ϩ /Fe).
Subunit interactions in ferritin occur between dimers and tetramers, as well as trimers (21)(22)(23)(24)(25)34). The assembly of H-L134P ferritin subunits into the typical supramolecular ferritin structure, except at the subunit trimer junctions, emphasizes the contributions of the subunit dimer and tetramer interactions to the structure (1, 2, 36); subunit trimer interactions contribute more to entry and exit of iron. Localized flexibility of ferritin at the subunit trimer junctions, caused by substitution of proline for conserved leucine 134, acts like a camera shutter increasing the aperture. Regulated iron release in vivo could result from cytoplasmic molecules causing similar conformational changes in ferritin.