Dynamics, stability and iron‐binding activity of frataxin clinical mutants

Abstract

Human frataxin is a mitochondrial protein whose deficiency is associated with the neurodegenerative disorder Friedreich ataxia (FRDA; OMIM 229300), a pathology characterized by neuronal death, cardiomyopathy and diabetes [1]. At the molecular level, the disease involves iron homeostasis deregulation and an impairment of the biosynthesis of iron-sulfur proteins [1][2][3][4]. The majority of FRDA patients (> 95%) are homozygous for a GAA repeat expansion within the first intron of the frataxin gene [5,6]. The expansion affects frataxin transcription, which results in a reduction of protein levels by 5-35%, depending on the insertion length. A small but significant number of FRDA patients are compound heterozygotes, Friedreich's ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the present study, we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations, G130V and D122Y, were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding (wild-type > W155R > I154F > D122Y > G130V), which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble ⁄ aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in the mutants; interestingly, these regions included those involved in iron-binding. In agreement, the iron metallochaperone activity of the Friedreich's ataxia mutants was affected: some mutants precipitate upon iron binding (I154F and W155R) and others have a lower binding stoichiometry (G130V and D122Y). Our results suggest that, in heterozygous patients, the development of Friedreich's ataxia may result from a combination of reduced efficiency of protein folding and accelerated degradation in vivo, leading to lower than normal concentrations of frataxin. This hypothesis also suggests that, although quite different from other neurodegenerative diseases involving toxic aggregation, Friedreich's ataxia could also be linked to a process of protein misfolding due to specific destabilization of frataxin.

The alteration of the C‐terminal region of human frataxin distorts its structural dynamics and function

“…8). As reported previously [14], the C-terminal domain of wild-type FXN exhibits rather uniform values of the relaxation rates along the protein sequence, a sign of a compact globular protein. Indeed, the <hnNOE> value is 0.81 AE 0.07, with only the C-terminus experiencing significantly lower values than average (Fig.…”

“…This increase in motions is globally distributed, suggesting a manifestation of cooperativity in dynamics. Protein dynamics of human FXN in the picosecond/ nanosecond range were previously studied by NMR [14,38]. Longitudinal and transverse relaxation rates and hnNOE indicated that FXN behaves as a rather rigid and compact globular protein with internal motions on the picosecond/nanosecond timescale at both termini.…”

“…However, some of them have been studied, showing differences in their thermodynamic stability (e.g. stability of wild-type > W155R > I154F > D122Y > G130V), a propensity to aggregate (mutants I154F and W155R precipitate upon iron binding) and a lower Fe 2+ binding stoichiometry (mutants D122Y and G130V) [13,14]. Interestingly, despite their decreased stabilities, NMR studies have shown that these clinical mutants retain a compact core and native-like dynamics [14].…”

“…stability of wild-type > W155R > I154F > D122Y > G130V), a propensity to aggregate (mutants I154F and W155R precipitate upon iron binding) and a lower Fe 2+ binding stoichiometry (mutants D122Y and G130V) [13,14]. Interestingly, despite their decreased stabilities, NMR studies have shown that these clinical mutants retain a compact core and native-like dynamics [14]. In addition, for the N146K, Q148R and R165C FRDA missense mutations, it was shown that the FXN function is compromised in the binding and activating of the SDUF complex (consisting of proteins NFS1, ISD11, ISCU2 and FXN) for Fe-S cluster biosynthesis [7].…”

The pathogenesis of Friedreich ataxia and the structure and function of frataxin

“…It is, however, unlikely that frataxins directly protect mitochondrial DNA, because their levels of expression would be insufficient for such a function. Some clinically occurring mutations have the effect of reducing thermodynamic stability with a consequent increasing likelihood of proteolytic digestion [30,41,42].…”