Deconstructing the role of key residues of the iron-binding motif EExxED using unstructured peptides and a grafted protein scaffold

VAZQUEZ, DIEGO SEBASTIAN; ARÁN, MARTÍN; SANTOS, JAVIER; AGUDELO, WILLIAM ARMANDO; GONZÁLEZ LEBRERO, MARIANO CAMILO; GIRAUDO, LAURA ROMINA; GONZÁLEZ FLECHA, F. LUIS

Congreso; IIILAFeBS, IX IberoAmerican congress of Biophysics, XLV SAB Annual Meeting; 2016; 2016

Proteins belonging frataxin family control intracellular iron homeostasis through a clusters of acidic residues that are apparently responsible for iron binding. To understand how frataxin works as an iron chaperon, we studied the interaction of metal ions with a putative iron binding site from the bacterial frataxin CyaY. For this purpose, the EExxED motif from the bacterial frataxin was grafted on a 16residue-long peptide, corresponding to the C-terminal α-helix from E. coli thioredoxin [1], giving the GRAP peptide [2]. We found by computational docking experiments that the motif has two mutually exclusive sites, involving only three acidic residues (ExxED or EExxE). In order to study the effect ofeach residue separately on the interaction with metal ions of different chemical nature (Mg2+, Al3+, Fe3+ and Ga3+), three engineered GRAP-derived peptides were synthesized carrying the sequences ExxED,EExxE and ExxE. Preliminary results obtained by circular dichroism suggest that: (i) In the absence of metal ions all the peptides behave as random-coil peptides at pH 7.0; (ii) Fe3+ and Al3+ are capable to induce/stabilize the helical structure of GRAP and peptide variants at pH 4.1 and pH 7.0, respectively; (iii) Nevertheless, Ga3+ only promote the acquisition of helical structure of GRAP, suggesting a fine tuning of the helix-coil transition. To gain further insights, the full length E. coli TRX was engineered including the EExxED motif in the C-terminal α-helix (TRXgrap), which is already structured in the native ensemble. By 15N-1H HSQC NMR experiments we found a specific interaction of Fe3+ with the EExxED motif causing the disappearance of cross-peak signals corresponding to residues located in the surrounding of the iron-binding motif.