Nanobodies as Novel Tools to Modulate Human Frataxin Stability and Function

Iron-sulfur clusters are essential cofactors for hundreds of proteins. In eukaryotic cells, the biogenesis of most iron-sulfur clusters occurs in the mitochondria and involves the Cys desulfurase supercomplex, which is activated by frataxin (FXN). The decrease of FXN expression, stability, and/or function results in Friedreich's ataxia (FA), a rare disease affecting 1 in 50,000 people. In this work, we propose modulating the conformational stability of FXN through nanobody interactions as a viable strategy to maintain FXN function. Several nanobodies specific to human FXN were selected via phage display, demonstrating a wide range of effects on Cys desulfurase activity. We focused on four nanobodies that exhibited strong interactions with FXN ( K _D = 1–30 nM) and stabilized the pathogenic FXN variant G130V by increasing its Tm by 15°C. The interaction between nanobodies and FXN was characterized using various biophysical tools, including NMR, SAXS, and X-ray diffraction. Three of the nanobodies bind to a similar region, and the structures of the corresponding nanobody-FXN complexes were solved by X-ray diffraction, showing a similar binding mode. In contrast, the fourth nanobody binds to alpha-helix 1, as determined by NMR and SAXS. The biological effects of nanobody expression were studied in human cells. The subcellular localization, effect on cell viability, Fe-S-dependent enzymatic activities, and oxygen consumption rates were analyzed. The expression of nanobodies sharing the same binding mode did not alter these key metabolic variables, suggesting that the interaction with FXN did not disrupt the pathway. Overall, these results suggest that nanobodies can be employed as tutor mitochondrial proteins to investigate the function modulation of unstable pathogenic FXN variants in FA models.