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

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

San Javier, Tucumán, Argentina

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 byneurological impairment and cardiomyopathy. It is caused by a deficiencyin the activity of frataxin (FXN), a mitochondrial iron chaperone thatregulates the transference of this metal ion to other proteins. Our previousresults indicate that the C-terminal region (CTR) is a crucial element in thestabilization of FXN. The presence of this stretch of residues enables thismacromolecule to smoothly modulate its stability and dynamics.1 Thesemotions may play a key role in the protein function given that the existenceof point mutations in this region leads to the development of FA.In this work we study the natural disease-associated mutant L198R, whichintroduces a voluminous residue and a positive charge in the apolarinteraction surface between the CTR and residues from both helices. Wesuggest that this mutation could produce changes in the dynamics of theCTR, probably affecting the stability and dynamics of the FXN core, as ithappens when the CTR is completely removed.1 In addition, mutant L198Awas studied as a model where native apolar interactions are weakened,without further perturbing the core. Besides, L198 was also mutated tocysteine as a tool to analyze local dynamics through the reactivity of theirthiol.Our results indicate that, while having a native-like topology, mutants arethermodynamically destabilized when compared with wild-type FXN, asshown by equilibrium unfolding experiments. Moreover, moleculardynamics simulations suggest that FXN L198R presents a more flexibleCTR and α unit than wild-type FXN. This prediction was analyzed andproven right by means of RMN experiments and limited proteolysis assays.All in all, our findings confirm that the stability of the native state is largelyperturbed by point mutation L198R, and that the functional deficiency ofthis mutant may be explained by an increased flexibility and thedestabilization of the interactions between the CTR and the α-helices.