Structural Dynamics and the Consolidation of Protein Function by Quaternary Addition of Nanobodies

MARÍA FLORENCIA PIGNATARO; MARIA FLORENCIA PAVAN; KARL ELLIOTH SEWELL; LORENA ARCE; DANIELA MILITELLO; HERNAN GENTILI; ITATÍ IBAÑEZ; JAVIER SANTOS

virtual

Congreso; Tercer Encuentro & Primer Workshop de la Red Argentina de Tecnología Enzimática (Red TEz); 2021

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 activator FXN. The supercomplex has a hetero decameric structure: (NFS1-ACP-ISD11-ISCU-FXN)2. The functional form of the L-Cys desulfurase NFS1 is stabilized by the interaction with ACP-ISD11. The Fe-S cluster biosynthesis involves (a) the desulfurase activity that uses L-Cys to produce L-Ala and a persulfur, and (b) iron, sulfur, and electron transferring to ISCU, for the Fe-S cluster assembly. Key residues of the FXN surface form part of the assembly site.Here, we propose the quaternary addition of nanobodies to the supercomplex to modulate its conformational stability and function. Nanobody libraries were prepared and several nanobodies showing an affinity for FXN were selected using the phage display technology. 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, a small number of nanobodies that bind to FXN did not alter the activity of the supercomplex. We will investigate whether these antibodies could contribute to successfully stabilize unstable FXN variants, improving Fe-S cluster biosynthesis. Furthermore, we think that these new tools will contribute to the understanding of this intricated catalytic process.