Frataxin Function and Quaternary Addition of Small Trojan Tutor Proteins

PIGNATARO, MARÍA FLORENCIA; HERRERA, MARÍA GEORGINA; CASTRO, IGNACIO HUGO; SEWELL, KARL ELLIOTH; ARAN, MARTÍN; NOGUERA, MARTÍN EZEQUIEL; SANTOS, JAVIER

Congreso; International Ataxia Research Conference; 2019

We propose the quaternary addition of a small tutor protein to modulate the frataxin (FXN) structural dynamics and the activity of the supercomplex involved in Fe-S cluster biosynthesis. We used Ribosome Display technology and the Sac7d protein as the scaffold to select affitins exhibiting the capability of interacting with human FXN. Two binders were obtained, Aff186 and Aff224. Affitins were prepared in E. coli and purified. The proteins are structured as judged by CD spectroscopy and temperature-induced unfolding experiments. Affitins showed a marked tendency to dimerize (SEC-FPLC). For both affitins, when DTT was absent, an intermolecular disulfide bond was formed, as detected by ESI-MS. Interaction between affitins and FXN was confirmed and investigated in detail in vitro using ELISA, SPR and ITC. Additionally, we were able to map the binding site of Aff186 and Aff224 on the region of the acidic ridge of FXN by analyzing chemical shift perturbations (NMR). The interaction between affitins and FRDA variants (G130V, L198R and W155R) or designed stable FXN mutants was also evaluated. Furthermore, we investigated the affitin-induced modulation of desulfurase enzymatic activity of the human supercomplex NFS1-ACP-ISD11-ISCUFXN. Moreover, we prepared a Trojan variant of Aff224 to deliver the affitin to the mitochondrial matrix to explore the effect of this molecule in the cellular environment. A new screening with a subsequent round of selection is being carried out to expand the diversity of affitins in order to obtain tutor proteins that can interact with FXN: (a) positively modulating FXN function in the context of the supercomplex; (b) maintaining FXN available to promoteactivation; or (c) stabilizing mutant variants of FXN for enough time to allow FXN-supercomplex interaction and activation.