Modulation of the Mitochondrial Iron-Sulfur Cluster Assembly Function by Nanobody-Frataxin Interaction

MARÍA FLORENCIA PIGNATARO; NATALIA BRENDA FERNÁNDEZ ; MARÍA FLORENCIA PAVÁN; ALBA GARAY; RAFAEL MOLINA; JULIÁN GROSSI; MARTÍN NOGUERA; ANTONELA VILA; HERNÁN GENTILI; NAIRA ANTONIA RODRÍGUEZ; MARTÍN ARAN; JUAN ANTONIO HERMOSO; LORENA ITATÍ IBAÑEZ; JAVIER SANTOS

Cordoba

Congreso; SAB; 2023

Sociedad Argentina de Biofísica

Modulation of the Mitochondrial Iron-Sulfur Cluster Assembly Function by Nanobody-FrataxinInteractionMaría Florencia Pignataro 1,2,4 , Natalia Brenda Fernández 1,2,4 , María Florencia Paván 3 , Alba Garay 5 , RafaelMolina 5 , Julián Grossi 1,2 , Martín Noguera 1,4 , Antonela Vila 1,2 , Hernán Gentili 1,2 , Naira Antonia Rodríguez 1,2 ,Martín Aran, Juan Antonio Hermoso Domínguez 6 , Lorena Itatí Ibañez 3, , Javier Santos 1,2,41 Instituto de Biociencias, Biotecnología y Biología Traslacional (iB3), Universidad de Buenos Aires, Buenos Aires, Argentina.2 Departamento de Fisiología, Biología Molecular y Celular (DFBMC), Facultad de Ciencias Exactas y Naturales (FCEN),Universidad de Buenos Aires (UBA), Buenos Aires, Argentina.3 Instituto de Química Física de los Materiales, Medio Ambiente y Energía (INQUIMAE), CONICET, FCEN, UBA, , CiudadUniversitaria, Buenos Aires, Argentina.4 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Godoy Cruz 2290 (C1425FQB) CABA – RepúblicaArgentina.5 Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física "Rocasolano", CSIC, Serrano 119, 28006-Madrid Spain.Friedreich Ataxia (FA) is a neurodegenerative disease caused by reduced levels of FXN (and/orunstable forms of this protein). The primary contributor to cellular dysfunction in FA is theimpairment of Iron-sulfur (Fe-S) cluster production. Fe-S clusters are essential cofactors foundin all known life forms, with hundreds of proteins reliant on these cofactors for their function.In eukaryotes, the majority of Fe-S cluster biogenesis occurs within the mitochondria and isfacilitated by the Iron Sulfur Cluster assembly machinery (ISC). This machinery consists of theNFS1-ISD11-ACP complex, which includes the cysteine desulfurase NFS1—an enzymeresponsible for desulfurizing L-Cys to produce L-Ala, along with a persulfur (R-S-SH), andfrataxin (FXN), which is necessary for efficient Fe-S cluster biogenesis. In this work, in order tomodulate FXN conformational stability and function, we propose the quaternary addition ofhumanized llama nanobodies (NB) targeting FXN. We obtained NB libraries and severalnanobodies showing an affinity for FXN were selected using phage display. The interaction wasfirstly evaluated by SEC-FLPC and NMR. The activation of Cys-desulfurase function by FXN wasevaluated in presence of NBs and a broad range of effects were found. Importantly, nanobodieswere able to stabilize a pathological FXN variant (G130V) in vitro. We obtained the dissociationconstants (Kd) for some FXN-NB complexes by Biolayer Interferometry (BLI) (Kds between 1-30nM). Moreover, to better understand the FXN modulation, the structure of the selected NB-FXNcomplexes was resolved by X-ray diffraction, which allowed us to map the complex on thestructure of the decameric supercomplex (NFS1/ACP-ISD11/ISCU/FXN) 2 . Importantly, the mostpromising NB were expressed in HEK 293T cells showing high levels of expression andmitochondrial localization. NB expression did not significantly alter Fe-S cluster dependentenzymatic activities and mitochondria oxygen consumption rates. We are presently examiningNB expression and functional modulation in FRDA cellular models to address the possibility ofrescuing FXN function. Additionaly, these novel tools will enhance our comprehension of thiscomplex catalytic process.Acknowledgements. This work was supported by Friedreich´s Ataxia Research Alliance (FARA), ANPCyTand CONICET