Unraveling of the E-helices and Disruption of 4-Fold Pores Are Associated with Iron Mishandling in a Mutant Ferritin Causing Neurodegeneration

Abstract

Mutations in the coding sequence of the ferritin light chain (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy, which is characterized by the presence of intracellular inclusion bodies containing the mutant FTL polypeptide and by abnormal accumulation of iron in the brain. Here, we describe the x-ray crystallographic structure and report functional studies of ferritin homopolymers formed from the mutant FTL polypeptide p.Phe167SerfsX26, which has a C terminus that is altered in amino acid sequence and length. The structure was determined and refined to 2.85 Å resolution and was very similar to the wild type between residues Ile-5 and Arg-154. However, instead of the E-helices normally present in wild type ferritin, the C-terminal sequences of all 24 mutant subunits showed substantial amounts of disorder, leading to multiple C-terminal polypeptide conformations and a large disruption of the normally tiny 4-fold axis pores. Functional studies underscored the importance of the mutant C-terminal sequence in iron-induced precipitation and revealed iron mishandling by soluble mutant FTL homopolymers in that only wild type incorporated iron when in direct competition in solution with mutant ferritin. Even without competition, the amount of iron incorporation over the first few minutes differed severalfold. Our data suggest that disruption at the 4-fold pores may lead to direct iron mishandling through attenuated iron incorporation by the soluble form of mutant ferritin and that the disordered C-terminal polypeptides may play a major role in iron-induced precipitation and formation of ferritin inclusion bodies in hereditary ferritinopathy.

Mutant Ferritin L-chains That Cause Neurodegeneration Act in a Dominant-negative Manner to Reduce Ferritin Iron Incorporation

“…We did not observe the difference in rate of iron oxidation between Lwt and L167fs described in Ref. 25, and under those conditions the kinetics at 310 nm of the two proteins were much slower and overlapping (not shown). The L-chains assist the H-ones in the mechanism of ferritin iron incorporation, thus their functionality must be analyzed when both are together in the same molecule.…”

“…Both samples migrated to a molecular mass of ϳ21 kDa with a very small quantity of truncated protein detected around 19 kDa. These data altogether suggest that, in the vicinity of the 4-fold molecular symmetry axes, the C-terminal part of L167fs is turned outside toward the solvent, as indicated before (25), leading to the disruption of the tightly packed hydrophobic pore, and to the formation of a large permeable opening. This disruption occurs also in heteropolymers in which a single mutant subunit is present in the 4-fold axes as in the models shown in Fig.…”

“…24 and 25 showing the massive precipitation of the mutant when incubated with 3000 -4000 irons/molecule. Baraibar et al (25) used a protein with an altered N terminus, which differs from the native one of our proteins, and this might affect protein solubility. More important, L-homopolymers lack the ferroxidase activity, which is necessary for in vivo iron incorporation (47), thus the in vitro conditions used to study their functionality are far from the physiological ones.…”

“…The mutant L154fs was shown to be poorly soluble, to coassemble with H-and L-ferritins, and to reduce the functionality of the ferritin heteropolymers (23). In contrast, the mutant L167fs was shown to be as soluble as L-wild type (Lwt), to be less thermostable, and to form aggregates when incubated with an excess of Fe(II) in aerobic conditions (22,24,25). It was proposed that these iron-rich ferritin aggregates contribute to the formation of ferritin bodies in vivo and alter ferritin functionality (26,27).…”

Iron, Aging, and Neurodegeneration

“…The disease has early and late onset forms and is associated with motor symptoms, spasticity and cognitive deficits [34]. The cause is usually a frameshift mutation in exon 4 for the L-ferritin gene, which causes a conformational change in the C-terminus of the molecule and alters its ability to store iron [35,36]. The mutation results in iron/ferritin-rich aggregates forming in cells [37].…”