LOOP ALTERATIONS AND THE CONSEQUENCES ON FOLDING, STABILITY AND STRUCTURAL DYNAMICS OF THE HUMAN FRATAXIN

NOGUERA MARTÍN EZEQUIEL; ARAN MARTÍN; GALLO MARIANA; SANTOS JAVIER

Roma

Congreso; XLIV National Congress on Magnetic Resonance; 2015

We study the structure, stability and internal motions of human frataxin (hFXN), a highly conserved mithocondrial protein involved in iron homeostasis and iron-sulphur cluster assembly, whose deficiency causes the neurodegenerative disease Friedreich?s ataxia. One of the pathological mutations that cause Friedreich?s ataxia is the early truncation of the protein producing a frataxin variant lacking of the C-terminal region (CTR). In a recent work, we found that the CTR of hFXN is crucial for consolidation of hFXN structure: deletion of the CTR destabilizes the global structure and alters the dynamics of the whole protein [1-2]. Molecular dynamics simulations and preliminary NMR experiments suggest that the mobility of the CTR and the loop-1 could be coupled. Sequence changes that alter the local dynamics of the CTR, also alter the dynamics of loop-1. The loop-1 is part of the region of frataxin involved in iron binding.In this work, we investigated the effect of loop-1 alterations on hFXN stability and dynamics. Loop-1 mutants were generated by site-directed mutagenesis, expressed and purified as the wild-type protein. Circular dichroism analysis, equilibrium unfolding experiments and light scattering measurements, indicated that loop-1 variants are well folded, stable and monomeric. This demonstrates that this structural element can be perturbed by mutations without inducing severe alterations in the tertiary structure or the aggregation state of the protein. However, a glycine-insertion variant exhibits higher sensitivity to protease action, suggesting an increase in mobility in the vicinity of loop-1. In this context, a complete NMR characterization of this variant indicates that even though fast motions are only locally increased upon loop-1 mutation, slower conformational motions of hFXN are significantly enhanced in regions that are not in direct contact with loop-1. Furhtermore, protection against proton-solvent exchange is also decreased for these regions in the loop-1 mutant. These results suggest a long-range relationship between loop-1 dynamics and motions of an extended region of the protein, far from the former, which includes residues from the beta-sheet and alpha-helix 1. References:[1] S.E. Faraj, E.A. Roman, M. Aran, M. Gallo, J. Santos FEBS J. 281, 3397-3419 (2014)[2] E.A. Roman, S.E. Faraj, M. Gallo, A.G. Salvay, D.U. Ferreiro, J. Santos PLoS One 7, e45743 (2012)