Frataxin Function and Quaternary Addition of Small Trojan Tutor Proteins

PIGNATARO, MARÍA FLORENCIA; SEWELL, KARL ELLIOTH; SANTOS, JAVIER; HERRERA, M.G; ARAN, MARTÍN; CASTRO, IGNACIO HUGO; NOGUERA, MARTÍN E.

Washington

Congreso; International Ataxia Research Congress; 2019

We propose the quaternary addition of a small tutor protein to modulate the frataxin (FXN) structural dynamicsand the activity of the supercomplex involved in Fe-S cluster biosynthesis. We used Ribosome Display technologyand the Sac7d protein as the scaffold to select affitins exhibiting the capability of interacting with human FXN. Twobinders were obtained, Aff186 and Aff224. Affitins were prepared in E. coli and purified. The proteins are structuredas judged by CD spectroscopy and temperature-induced unfolding experiments. Affitins showed a marked tendencyto dimerize (SEC-FPLC). For both affitins, when DTT was absent, an intermolecular disulfide bond was formed, asdetected by ESI-MS. Interaction between affitins and FXN was confirmed and investigated in detail in vitro usingELISA, SPR and ITC. Additionally, we were able to map the binding site of Aff186 and Aff224 on the region of the acidicridge 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 theaffitin-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 explorethe effect of this molecule in the cellular environment. A new screening with a subsequent round of selection isbeing 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 andactivation.