Helix-Coil Transition Induced by Metal Ion Interaction with a Grafted Iron Binding Site of the CyaY Protein Family

DIEGO S. VAZQUEZ; WILLIAM A. AGUDELO; ANGEL YONE; NORA VIZIOLI; MARTÍN ARÁN; F. LUIS GONZÁLEZ FLECHA; MARIANO C. GONZÁLEZ LEBRERO; JAVIER SANTOS

DALTON TRANSACTIONS

ROYAL SOC CHEMISTRY

Lugar: CAMBRIDGE; Año: 2014 vol. 44 p. 2370 - 2379

1477-9226

Iron-protein interactions are involved in electron transfer reactions. Alterations of these processes are present in a number of human pathologies; among then, in Friedreich´s ataxia, in which a deficiency in functional frataxin, an iron-binding protein, leads to the progressiveneuromuscular degenerative disease. The putative iron-binding motif of acidic residues EExxED was selected from the first α-helical stretch of the frataxin protein family and grafted on a foreign peptide scaffold corresponding to the C-terminal α-helix from E. colithioredoxin. The resultant grafted peptide named GRAP was studied applying experimental (circular dichroism, isothermal titration calorimetry, capillary zone electrophoresis, thermaldenaturation, NMR) and computational approaches (docking, molecular dynamics simulations). Although isolated GRAP lacks a stable secondary structure in solution, when iron is added, the peptide acquires an α?helical structure. Here we have shown that the designedpeptide is able to specifically bind Fe3+ with moderate affinity (KD= 1.9±0.2 μM) and a 1:1 stoichiometry. Remarkably, GRAP/Fe3+ interaction is entropically driven (ΔH°=-1.53 ± 0.03 kcal mol-1 and TΔS° =6.26 kcal mol-1). Experiments and simulations indicate that Fe3+ interactswith the peptide through three acidic side chains, inducing the α-helical conformation of the grafted motif. In addition, the acidic side chains involved undergo significant conformational rearrangements upon binding, as judged by the analysis of MDs. Altogether, these resultscontribute to an understanding of the iron-binding mechanisms in proteins and, in particular, in the case of human frataxin