Preliminary Structural Characterization of an Iron-binder Engineered Thioredoxin.

VAZQUEZ DS; ARÁN M; SANTOS J

Congreso; Reunión anual de la SAB; 2014

Frataxin (FXN), a member of the CyaY protein family, is characterized by the presence of a large cluster of acidic residues on its surface. FXN is principally involved in iron-sulfur cluster biogenesis, iron binding and exchange to other proteins. We have designed and characterized experimentally and computationally a short peptide (called GRAP) that includes the EExxED motif from the CyaY protein family, using the sequence of the C-terminal helix of E. coli thioredoxin (TRX) as backbone scaffold. GRAP of sequence LSKGQLEEFLEDNLAY shows certain specificity for Fe2+ stoichiometry and a KD of 1.9±0.2 μM. Iron binding is an entropically driven process that it is guided more likely by changes in hydration. Both binding and peptide folding processes take place upon metal interaction. Subsequently, to uncouple the coil-to-helix transition to study both the iron-binding affinity and the effect on intrinsic structural dynamics, we grafted the motif onto the full-length TRX (TRXgrap), a context that more accurately mimics the environment of the iron binding site in a real protein. TRXgra protein is structured and the EExxED grafting showed a marginal impact on its global stability (ΔΔGN↔U ≈ 1 kcal mol-1 activity (50%). Iron binding experiments indicated a preliminary stoichiometry of 1:1. To characterize at the residue level the TRXgrap Fe3+ theoretical experiments were carried out. The evaluation of 1H-15N HSQC spectra of TRXgrap in the presence or absence of iron shows that the interaction causes the disappearance of signals of some residues located in the surrounding of the iron-binding motif, compatible with the paramagnetic properties of this metal ion. On the other hand, molecular dynamic simulations of the apo and holo forms shows significant perturbations at the active site in the reduced state suggesting effects of mutation and metal binding at medium-large distances and explaining the observed reduction of enzimatic activity. Taken as a whole, these results open the possibility to investigate the effect of protein-metal interaction in structural and folding dynamics. ). Noteworthy, apo form exhibits a diminished oxidoreductase