Modulation of the Structural and Functional Dynamics of the L-Cys Desulfurase Supercomplex by Nanobody Interactions

MARÍA FLORENCIA PIGNATARO; MARÍA FLORENCIA PAVAN; NATALIA BRENDA FERNÁNDEZ; NAIRA ANTONIA RODRÍGUEZ; JULIÁN GROSSI; HERNAN GUSTAVO GENTILI; LORENA PAOLA ARCE; DANIELA AYELÉN MILITELLO; KARL ELLIOTH SEWELL; MARTÍN ARAN; ITATÍ IBAÑEZ; JAVIER SANTOS

Rosario

Congreso; Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

Background and objectives: Iron-sulfur (Fe-S) clusters are essential cofactors present in all known forms of life, and hundreds of proteins require such cofactors to work. In eukaryotes, the biogenesis of most Fe-S clusters occurs in the mitochondria. The process involves the interactions and activities of several key proteins that form a supercomplex, namely L-Cys desulfurase NFS1, the heterodimer APC-ISD11, the scaffolding subunit ISCU, and the kinetic activator FXN. The supercomplex has a hetero decameric structure: (NFS1-ACP-ISD11-ISCU-FXN)2. The biosynthesis involves (a) the desulfurase activity that uses L-Cys to produce L-Ala and a persulfur (R-S-SH), and (b) iron, sulfur, and electron transferring to ISCU, for the Fe-S cluster assembly. Here, we propose the quaternary addition of nanobodies to the supercomplex to modulate its conformational stability and function. Methods: Nanobody libraries were prepared and several nanobodies showing an affinity for FXN were selected using the phage display technology. The interaction was evaluated by SEC-FLPC, fluorescence, ELISA and NMR. The function was studied by the methylene blue method and NMR. The stability was evaluated by Sypro-orange fluorescence. Results: Some nanobodies exhibited marked inhibitory activity of L-Cys-NFS1 desulfurase function. We suggest that these nanobodies may interact with FXN surfaces essential for the consolidation of the supercomplex. On the other hand, some nanobodies that bind to FXN showed only a slight inhibitory effect on the activity of the supercomplex. Nanobodies were able to stabilize an altered FXN variant (G130V). The binding site of some nanobodies was determined by NMR and compared to that predicted by Alpha fold 2. Conclusions: We are investigating whether these antibodies could successfully stabilize unstable FXN variants in the context of the supercomplex, improving Fe-S cluster biosynthesis in the cellular environment. Furthermore, we think that these new tools will contribute to the understanding of this intricated catalytic process.