Friedreich's ataxia-associated FXN L198R mutant exhibits diminished stability and altered dynamics

SANTIAGO E. FARAJ; ERNESTO A. ROMAN; MARTÍN ARÁN; MARIANA GALLO; JAVIER SANTOS

San Javier, Tucumán

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

Friedreich's ataxia (FA) is a progressive hereditary disease, characterized by neurological impairment and cardiomyopathy. It is caused by a deficiency in the activity of frataxin (FXN), a mitochondrial iron chaperone that regulates the transference of this metal ion to other proteins. Our previous results indicate that the C-terminal region (CTR) is a crucial element in the stabilization of FXN. The presence of this stretch of residues enables this macromolecule to smoothly modulate its stability and dynamics.1 These motions may play a key role in the protein function given that the existence of point mutations in this region leads to the development of FA. In this work we study the natural disease-associated mutant L198R, which introduces a voluminous residue and a positive charge in the apolar interaction surface between the CTR and residues from both helices. We suggest that this mutation could produce changes in the dynamics of the CTR, probably affecting the stability and dynamics of the FXN core, as it happens when the CTR is completely removed.1 In addition, mutant L198A was studied as a model where native apolar interactions are weakened, without further perturbing the core. Besides, L198 was also mutated to cysteine as a tool to analyze local dynamics through the reactivity of their thiol. Our results indicate that, while having a native-like topology, mutants are thermodynamically destabilized when compared with wild-type FXN, as shown by equilibrium unfolding experiments. Moreover, molecular dynamics simulations suggest that FXN L198R presents a more flexible CTR and α unit than wild-type FXN. This prediction was analyzed and proven right by means of RMN experiments and limited proteolysis assays. All in all, our findings confirm that the stability of the native state is largely perturbed by point mutation L198R, and that the functional deficiency of this mutant may be explained by an increased flexibility and the destabilization of the interactions between the CTR and the α-helices.