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; LORENA ITATÍ IBAÑEZ; JAVIER SANTOS

Rosario Santa Fe

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

SAB

Background and objectives: Iron-sulfur (Fe-S) clusters are essential cofactors present inall known forms of life, and hundreds of proteins require such cofactors to work. Ineukaryotes, the biogenesis of most Fe-S clusters occurs in the mitochondria. The processinvolves the interactions and activities of several key proteins that form a supercomplex,namely L-Cys desulfurase NFS1, the heterodimer APC-ISD11, the scaffolding subunitISCU, 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 thatuses L-Cys to produce L-Ala and a persulfur (R-S-SH), and (b) iron, sulfur, and electrontransferring to ISCU, for the Fe-S cluster assembly. Here, we propose the quaternaryaddition of nanobodies to the supercomplex to modulate its conformational stability andfunction.Methods: Nanobody libraries were prepared and several nanobodies showing an affinityfor FXN were selected using the phage display technology. The interaction was evaluatedby SEC-FLPC, fluorescence, ELISA, and NMR. The function was studied by the methyleneblue method and NMR. The stability was evaluated by Sypro-orange fluorescence.Results: Some nanobodies exhibited marked inhibitory activity of L-Cys-NFS1desulfurase function. We suggest that these nanobodies may interact with FXN surfacesessential for the consolidation of the supercomplex. On the other hand, some nanobodiesthat bind to FXN showed only a slight inhibitory effect on the activity of thesupercomplex. Nanobodies were able to stabilize an altered FXN variant (G130V). Thebinding site of some nanobodies was determined by NMR and compared to that predictedby Alpha fold 2.Conclusions: We are investigating whether these antibodies could successfully stabilizeunstable FXN variants in the context of the supercomplex, improving Fe-S clusterbiosynthesis in the cellular environment. Furthermore, we think that these new tools willcontribute to the understanding of this intricated catalytic process.