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

DIEGO S. VAZQUEZ; GIRAUDO LAURA; AGUDELO, WILLIAM A.; ARAN, MARTÍN; GONZALEZ-FLECHA, LF; MARIANO GONZALEZ LEBRERO; JAVIER SANTOS

Tucumán

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

SAB

Deconstructing the role of key residues of the iron-binding motif EExxED using unstructuredpeptides and a grafted protein scaffoldVazquez, Diego Sebastian1; Giraudo, Laura Romina1; Agudelo, William Armando2; Aran, Martín3;González Flecha, F. Luis1; González Lebrero, Mariano Camilo2; and Santos, Javier1,§.1Instituto de Química y Fisicoquímica Biológicas, Departamento de Química Biológica, Universidad deBuenos Aires and CONICET, Argentina. 2Departamento de Química Inorgánica, Analítica y QuímicaFísica, FCEN, UBA, INQUIMAE-CONICET, Argentina. 3Fundación Instituto Leloir and IIBBACONICET,Argentina. §Contact e-mail: javiersantosw@gmail.comProteins belonging frataxin family control intracellular iron homeostasis through a clusters of acidicresidues, that are apparently responsible for iron binding. To understand how frataxin works as an ironchaperon, we studied the interaction of metal ions with a putative iron binding site from the bacterialfrataxin 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 theGRAP peptide [2]. We found by computational docking experiments that the motif has two mutuallyexclusive 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 ofmetal ions all the peptides behave as random-coil peptides at pH 7.0; (ii) Fe3+ and Al3+ are capable toinduce/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 finetuningof the helix-coil transition. To gain further insights, the full length E. coli TRX was engineeredincluding the EExxED motif in the C-terminal α-helix (TRXgrap), which is already structured in thenative ensemble. By 15N-1H HSQC NMR experiments we found a specific interaction of Fe3+ with theEExxED motif causing the disappearance of cross-peak signals corresponding to residues located inthe surrounding of the iron-binding motif.Referencias1 Vazquez DS et al., The E. coli thioredoxin folding mechanism: The key role of the C-terminal helix,Biochimica et Biophysica Acta, 1854(2):127-137, 2015.2 Vazquez DS et al., A helix-coil transition induced by the metal ion interaction with a grafted ironbindingsite of the CyaY protein family, Dalton Transactions, 44, 2370-2379, 2015.AgradecimientosThis work was supported by grants from UBACyT, ANPCyT, and CONICET