Nanobodies as Novel Tools to Modulate Human Frataxin Stability and Function

Nanobodies as Novel Tools to Modulate Human Frataxin Stability and Function | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nanobodies as Novel Tools to Modulate Human Frataxin Stability and Function María Pignataro, Natalia Fernández, Alba Garay-Alvarez, María Pavan, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6122246/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Biophysics/Molecular biophysics/Supramolecular assembly Biological sciences/Biotechnology/Molecular engineering/Protein design Biological sciences/Structural biology/X-ray crystallography Iron-Sulfur Cluster Assembly Frataxin Friedreich Ataxia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 INTRODUCTION The study at the molecular level of iron-sulfur cluster biosynthesis has recently increased the comprehension of some of the relationships between structure and function concerning the mitochondrial supercomplex involved in this pathway. The Fe-S clusters are at the core of many essential biological activities. Mitochondrial enzymatic functions such as NADH dehydrogenase, aconitase, succinate dehydrogenase, and complex III from the respiratory chain, lipoic acid synthetase, and processes such as nuclear DNA repair [ 1 ], the chemical modifications of transfer RNAs that affect base recognition between codon and anticodon [ 2 ], and the cytoplasmic Fe-S cluster assembly by the cytosolic iron-sulfur cluster assembly (CIA) system require the mitochondrial cysteine desulfurase supercomplex [ 3 , 4 ]. This supercomplex consists of several proteins, all of them encoded by nuclear DNA (Fig. 1) [ 5 ]. NFS1 cysteine desulfurase is one of the essential enzymes. It is translated as a 50 kDa precursor, imported into the mitochondrial matrix, and processed to a mature dimeric form (89 kDa) [ 6 , 7 ]. This enzyme is a dynamic hub that collects many other proteins on its surface. The L-Cys desulfurization reaction depends on pyridoxal phosphate (PLP), and although it is multifaceted, it can be summarized in a series of key stages [ 8 ]: a) formation of the outer Schiff base with the L-Cys substrate; b) formation of the persulfide-enzyme intermediate; c) release of L-Ala and transfer of the persulfide to a Cys in a mobile loop of the enzyme (Cys381 in the case of human NFS1); and d) the subsequent transfer of the -SH group to the iron-sulfur cluster assembly enzyme (ISCU2). In subsequent reactions, the persulfide groups will be reduced and combined with iron to form the Fe-S clusters. ISCU2 is an essential scaffold protein that supports the assembly of the [2Fe-2S] cluster at its active site, which is composed of two cysteines (Cys95 and Cys138), an aspartic acid (Asp71), and one histidine (His137) [ 9 , 10 ]. Crystallographic data show that the ISCU2 active site is in close contact with the Cys-loop of the NFS1, where Cys381 is located [ 11 ]. It was demonstrated that the L-cysteine increases the binding efficiency of human ISCU2 to NFS1 nine-fold ( K D = 0.2 µM with cysteine and 1.7 µM without [ 11 ]. Downstream, trafficking proteins and targeting factors, including a dedicated system of chaperones and co-chaperones, transfer the Fe-S cluster to acceptor proteins (e.g., enzymes) [ 12 – 14 ]. Another essential protein that forms the supercomplex is frataxin (FXN), which is the kinetic activator of the NFS1 and accelerates the persulfide transfer [ 15 ]. FXN is imported into the mitochondrial matrix as a 210-residue precursor and processed in two steps; the first produces an intermediate form (residues 41 to 210) and after that, the second stage produces the mature FXN form 81–210 (14.2 kDa) [ 16 , 17 ]. Current structural (Cryo-EM) and functional studies suggest that FXN simultaneously interacts with ISCU2 and with both subunits of the NFS1 dimer [ 5 ]. Recently, it was shown that the natural source of electrons is provided by the ferredoxin system involving NADPH, FdxR, and FDX2 [ 18 – 22 ]. Since FXN binds iron through its acidic region involving alpha helix 1, loop 1, and beta-strand 1, with a moderate K D [ 23 , 24 ], it is possible that this protein could also participate in the reaction delivering the Fe(II) metal ions to ISCU2, a process necessary for the biosynthesis of the clusters. Current experimental evidence indicates that FXN binds to iron in the context of the protein complex and that FXN could release the metal ion by transferring it to ISCU2 in the presence of L-Cys (the substrate) and the natural reducing agent FDX2 [ 19 ]. However, how the iron transfer to ISCU occurs remains unclear. It was shown that NFS1 is stabilized in its active dimeric form by a small protein called ISD11 (10.5 kDa), which also prevents NFS1 aggregation. ISD11 is an essential protein in eukaryotes [ 25 , 26 ], and it is completely absent in Bacteria and Archaea [ 25 ]. Furthermore, the alterations in the expression or structural dynamics of the proteins that conform to the supercomplex decrease the functionality of the pathway. Notably, decreased expression or function of ISCU2 results in the severe disease ISCU myopathy [ 27 , 28 ]. A pathogenic ISD11 variant (R68L) has been associated with the development of a mitochondrial genetic disorder, COXPD19. This mutation resulted in the reduction of the affinity between ISD11 and NFS1, increasing the tendency of NFS1 to aggregate and the accumulation of reactive oxygen species, leading to mitochondrial dysfunction [ 17 ]. Besides, mutation R72Q of NFS1 was described as the cause of the deficit in NFS1 function, which results in the infantile mitochondrial complex II/III deficiency, an autosomal recessive mitochondrial disease that is characterized by lactic acidemia, respiratory chain complex II and III deficiency, and abnormal mitochondria [ 29 ]. In particular, the decrease of FXN expression results in Friedreich's ataxia (FA), an inherited neurodegenerative rare disease affecting 1 in 50,000 people [ 30 ]. In this context, one of our main research goals is to select specific small tutor proteins that may provide conformational stability to pathogenic, highly unstable variants of FXN while preserving key interactions with other proteins of the mitochondrial supercomplex. We have named this strategy quaternary addition [ 38 ]. Recently, we selected affi_224, a synthetic FXN binder, employing the Ribosome Display strategy and archaeal Sac7D protein as the scaffold (Affitins) [ 38 ]. We showed that Affi_224, fused to a human mitochondrial targeting sequence, was successfully expressed in HEK-293T cell lines and interacted with FXN, as judged by co-immunoprecipitation. Moreover, when the wild-type FXN is replaced by the G130V variant, the presence of affi_224 increases the activity of the supercomplex in vitro . However, the latter did not occur when the wild-type FXN was replaced by the highly unstable W173G variant. In this sense, other in vitro experiments showed that Affi_224 did not form a stable complex with FXN. Furthermore, affi_224 is an unstable protein per se , and it tends to form dimers. These features might preclude its use in further studies. With this background, we explored other scaffolds for developing FXN tutors to investigate the stabilization strategy and chose the camelid-derived nanobody scaffold (V HH or NBs). Highly specific NBs can be obtained by phage display strategy from an RNA library of camelid antibody sequences prepared after a convenient immunization of the camelid with the target protein. Camelid heavy chain antibodies are formed by two heavy chains, each one containing a variable domain (V HH ), which includes three complementarity-determining regions (CDR1, CDR2, and CDR3, the latter longer than the others) (Fig. 1 D ). NBs are small (15kDa) and conformationally stable proteins [ 39 ] that can be easily and cheaply produced compared to conventional monoclonal antibodies. NBs can be expressed in E. coli . In addition, their small size may allow better tissue penetration [ 40 ]. Even though NBs usually display low immunogenicity, they can be humanized to reduce this characteristic and, therefore, be suitable for therapeutic applications in humans [ 41 ]. NBs can be expressed by mammalian cells as "intrabodies" while retaining their conformational stability. In particular, an advantage of NBs is that they can be delivered by gene therapy as single genes; these proteins are currently being used to explore treatments for various neurodegenerative disorders and cancer disease, and some of them are in clinical trials [ 39 , 42 , 43 ]. In this paper, we selected more than twenty FXN-specific NBs. The interaction between FXN and four of them was deeply investigated in vitro and in a cellular environment. MATERIALS AND METHODS DNA Sequencing, Protein Concentration, and Mass Spectrometry Analysis Identity was verified by DNA sequencing using the Macrogen facility. Absorption spectra were obtained with a JASCO V-730 BIO spectrophotometer (Tokyo, Japan). Protein concentration was determined using the extinction coefficient obtained from the amino acid sequence using the ProtParam tool from ExPASy. Protein purity was estimated by SDS-PAGE analysis, and the analysis of the intact masses was performed by mass spectrometry in the National Laboratory of Research and Services in Peptides and Proteins using an LCQ DUO ESI ion trap (Thermo Finnigan) or QExactive Orbitrap (Thermo Scientific) spectrometers. HPLC Analysis of Protein Samples Reverse phase HPLC analysis was performed using a JASCO system equipped with an autoinjector, UV detector, and a thermostatic oven at 25 °C. Gradients from 0 to 100% acetonitrile were done, and 0.05% TFA ( v/v ) were added to the solvents and samples. The columns used were analytical C18 (Higgins Analytical, Inc. U.S.A.), and the flow was 1.0 mL min − 1 . Proteins were monitored at 220 nm. Before injection, all samples were centrifuged. Expression and Purification of the Human Supercomplex Core (NFS1/ACP-ISD11) The DNA sequences of the human mature form of the human cysteine desulfurase NFS1 enzyme (NFS1Δ55), the ISD11, and the human mitochondrial acyl carrier protein (ACP, the mature form) were optimized for protein expression in E. coli BL21 (DE3) by BIO BASIC Inc (Markham ON, Canada). NFS1Δ55 and ISD11 were cloned in a pETDuet-1 plasmid, whereas ACP was cloned in a pACYCDuet-1 for co-expression. NFS1 amino acid sequence included the RSGHHHHHH tag in the N-terminal for purification and antibody recognition. Protein expression was induced by 1mM IPTG when the bacterial culture reached OD 600nm = 1.0. Co-expression was carried out overnight at 20°C (at 250 rpm). The purification of the complex (NFS1/ACP-ISD11) 2 was performed from the soluble fraction of E. coli BL21 DE3 cultures using a Ni 2+ -NTA-agarose column (first step). The protein was eluted with 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0. After that, 2 mM DTT was added and the protein was dialyzed overnight against the same buffer, without imidazole, at 4°C. Afterwards, it was frozen at -70°C. This protocol minimized aggregation and oxidation of the protein. Before each experiment, (NFS1/ACP-ISD11) 2 was thawed in ice and centrifuged. Protein concentration was determined spectroscopically using an absorption coefficient ε 280nm = 53750 M –1 cm –1 . Expression and Purification of ISCU2 The ISCU2 DNA sequence corresponding to the mature form was optimized for E. coli overexpression by Explora Biotech (Rome, Italy) and subcloned in a pE22b plasmid, with a C-terminal His6 tag. Protein induction was carried out by adding 1mM IPTG final concentration (3h, 37°C and 250 rpm). ISCU2 was purified using a Ni 2+ -NTA-agarose column equilibrated with 20 mM Tris-HCl, 300 mM NaCl, pH 7.5. The elution was performed with 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 7.5. The collected elution fractions were analyzed by SDS-PAGE and pooled. After that, an extensive dialysis step was performed (4 °C, in a 20 mM Tris-HCl, 300 mM NaCl, pH 7.5, 1 mM DTT). Protein purity was > 95%, as evaluated in SDS-PAGE. No aggregation was observed, as inferred from size exclusion chromatography (SEC) analysis. The protein was stored at − 70 °C until use. Protein concentration was determined spectroscopically using an absorption coefficient ε 280nm = 11,460 M –1 cm –1 (1 mg/mL protein solution represents Abs 280nm = 0.70). The zinc content in ISCU samples was evaluated by atomic absorption spectroscopy at the Department of Bromatology, College of Pharmacy and Biochemistry, Universidad de Buenos Aires . Zinc concentration in ISCU2 preparations was 0.14: 1 (zinc: ISCU2, molar ratio). Expression and Purification of FXN Human FXN (residues 90–210) was overexpressed and purified as previously described for the wild-type protein [ 34 ]. Briefly, bacteria cultures ( E. coli BL21 (DE3), 2–3 L Terrific Broth, pH 7.2) were grown at 37 °C and 280 rpm. Protein expression was induced at DO = 0.8-1.0 with 1.0 mM IPTG. After induction (3.5 hours), bacteria were centrifuged (6,000 rpm), and the pellet was stored at − 20 °C until cell disruption by sonication (in an ice-water bath). The soluble fraction was separated by centrifugation (10,000 rpm, 30 min). The soluble fraction was incubated with 10 mM EDTA and loaded onto an ion exchange chromatography (DEAE DE52 matrix). The protein was eluted with a 300 mL linear gradient from 0.0 to 1.0 M NaCl (the buffer was 20 mM Tris-HCl, 1 mM EDTA, pH 7.0). Fractions with FXN (identified by SDS-PAGE) were loaded onto a Sephadex G–100 column (SEC, 93 cm × 62.7 cm, equilibrated with 20 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, pH 7.0). FXN concentration was determined spectroscopically using an absorption coefficient ε 280nm = 26,930 M –1 cm –1 (1 mg/mL protein solution represents Abs 280nm = 2.00). Purity was > 98% as evaluated in SDS-PAGE. NFS1 Cysteine Desulfurase Activity For enzymatic desulfurization of L-Cys to L-Ala and sulfide by the (NFS1/ACP-ISD11/ISCU/FXN) 2 supercomplex, concentrations of proteins, substrate and the reducing agent DTT were set according to a previous paper by Tsai and Barondeau [ 44 ]. Reactions contained 1.0 µM NFS1/ACP-ISD11, 3.0 µM ISCU, and 1.0 µM FXN, and samples were supplemented with 10 µM PLP, 2.0 mM DTT and 1.0 µM FeSO 4 (final concentrations). In all cases, the reaction buffer was 50 mM Tris-HCl and 200 mM NaCl, pH 8.0, and reactions were started by the addition of 1.0 mM L-Cys (or variable L-Cys concentrations, as we describe below). Samples were incubated at room temperature (25°C) for 30 min. For the reactions including NBs, each NB was preincubated with FXN (10 min), and then, both proteins were added to the reaction mix. The Methylene Blue Method Sulfide was determined by the methylene blue method [ 44 , 45 ]. For this analysis, H 2 S production was stopped by adding 50 µL of 20 mM N,N -dimethyl p- phenylenediamine in 7.2 M HCl and 50 µL of 30 mM FeCl 3 (prepared in 1.2 M HCl). Under these conditions, the production of methylene blue took 20 min. After that, samples were centrifuged for 5 min at 12000 x g , and the supernatant was separated. Absorbance at 670 nm was measured. Llama Immunization and Library Construction A llama located at INTA´s Camelids Experimental Unit was immunized intramuscularly with 150 µg per dose of human FXN 90–210 recombinant protein on days 0, 14, 28 and 50. Complete Freund’s adjuvant was used for the first dose, and incomplete Freund’s adjuvant for the following boosts. Antibody responses were monitored by ELISA on serum samples taken before each immunization. Llama inoculation and sample collection were conducted by trained staff, the study was approved by the Animal Care and Use Committee of INTA (CICUAE) under the protocol N° FR6.2-3/2020. Four days after the last boost, 150 mL of anticoagulated blood was used to isolate lymphocytes by Ficoll Paque Plus (GE Life Sciences, 17-1440-02) filled Leucosep tubes (GBO, 227290), total RNA was extracted (RNAeasy Midi, Qiagen 75144) and cDNA was prepared with oligo(dT) primers (First Strand cDNA Synthesis Kit, Roche 04379012001). VH and VHH genes were amplified with CALL001 (5’-GTCCTGGCTGCTCTTCTACAAGG-3’) and CALL002 (5’-GGTACGTGCTGTTGAACTGTTCC-3’) primers. A PCR amplicon of 0.7 kb was purified from gel (Wizard® SV Gel and PCR Clean-Up System, Promega A9282) and then used as a template in a nested PCR to specifically amplify the VHH fragments using VHH-BACK-SAPI (5’-CTTGGCTCTTCTGTGCAGCTGCAGGAGTCTGGRGGAGG-3’) and VHH-FORWARD-SAPI (5’-TGATGCTCTTCCGCTGAGGAGACGGTGACCTGGGT-3’) primers. A Golden Gate assembly was done to clone the VHH sequences between two SapI sites of the phagemid vector pMECS-GG, following a protocol previously described [ 46 ]. Electro-competent E. coli TG1 cells (Lucigen 60502-1) were transformed with the purified ligation mixture and plated on a selective agar medium. A library of 1.8⋅10 9 individual transformants was obtained [ 47 ]. Selection of FXN Specific NBs To produce recombinant specific NBs, 1 mL of bacteria from the stock library was grown in 2⋅TY until OD 600nm = 0.6, afterward, VCS M13 helper phage (Stratagene, 200251) was used to infect exponentially growing bacteria. The resulting NB phage display library was panned three times on microtiter plates (Maxisorp Nunc) coated overnight at 4°C with 10 µg of each recombinant protein in 100 µL of PBS and 100 µL of PBS for the negative controls. The next day, wells were washed with PBST (PBS + 0.05% Tween 20) and blocked with 2% skim milk in PBST. Approximately 1⋅10 12 phage particles were preincubated with 10 µL of blocking solution in 100 µL of PBS for 30 min at room temperature by head-over-head rotation, then added on positive and negative wells and incubated for two hours on a vibrating platform (500 rpm.). During the first panning round, wells were washed 10 times with PBST, while wells were washed 20 and 25 times for the second and third rounds, respectively; 5 min incubation on a vibrating platform (300 rpm) was done every five washes. Specific phage particles were eluted with 0.25 mg/mL trypsin solution (Sigma-Aldrich, T1426) for 30 min followed by neutralization with 4 mg/mL AEBSF solution (Carl Roth, 2931.3). A second elution step was done by adding exponentially growing E. coli TG1 cells to positive and negative wells that were then incubated for 30 min at 37°C. Trypsin-eluted phage particles were amplified by infection of exponentially growing E. coli TG1 cells and later superinfected with VCS M13 helper phage. Phage particles obtained after both elution strategies were purified using PEG 6,000/NaCl precipitation and used for the next selection round. To obtain specific binders, individual TG1 colonies were screened by ELISA using periplasmic extract. For this, 95 colonies from the positive wells (different panning rounds and elution strategies) and 1 colony from the negative well were inoculated in 1 mL of 2⋅TY medium containing 100 µg/mL ampicillin and 0.1% glucose in a deep well plate. NB expression was induced after bacteria incubation for 3 h at 37°C and 200 rpm. with 1 mM IPTG. After 4 h induction, bacterial cultures were centrifuged, the pellets were frozen and thawed twice to disrupt cells and resuspended in 120 µL of PBS. On the other hand, the periplasmic extract (see below, NB purification) was used to determine the specificity of NB binding and to study their functional activity by the Methylene Blue method. Screening for Antigen Binders For screening experiments, the study of NB binding to FXN was conducted by ELISA. For this, microtiter plates (Maxisorp, Nunc) were coated overnight at 4°C with 200 ng/well of recombinant FXN or an irrelevant protein as negative controls, diluted in PBS. After three washes with PBST, wells were blocked with 3% skim milk in PBST, and 50 µL of the periplasmic extract was added to each well and incubated at room temperature for 2 h. After washing with PBST to remove excess of NB, specific binding was detected with horseradish peroxidase (HRP)-linked anti-HA antibody (Abcam, ab1190) diluted 1:1500. Finally, 50 µL of TMB substrate (3,3', 5,5' tetramethylbenzidine, BD 555214) was added. Absorbance at 450 nm was measured using an ELISA reader (TECAN). Nanobody Production and Purification A DNA sequence corresponding to the NB, preceded by a signal peptide for export to the periplasm (MKYLLPTAAAGLLLLAAQPAMA), C-terminal hemagglutinin (YPYDVPDY), and His (HHHHHH) tags, was cloned in an expression vector (pMECS), and E. coli WK6 was transfected with these constructions. Different expression protocols were carried out (temperature was modified from 20 to 37 °C, and expression was performed for 4 h or overnight). Some differences concerning yield and purity were observed for specific NBs. However, we chose to follow the same protocol for all the NBs. Protein induction was carried out by adding 1.0 mM IPTG final concentration (4h, 37°C and 190 rpm). Periplasm fluid was recovered by an osmotic shock. The pellet from 2 L of WK6 cell culture was resuspended in 30 mL of TES buffer (100 mM Tris–HCl pH 8.0, 1 mM EDTA, and 20% sucrose) pre-chilled in an ice-water bath. The resuspended cells were incubated for 60 min. After that, 90 mL of pre-chilled deionized water was added, and cells were incubated overnight. Both incubations were carried out on an ice-water bath in a rocking shaker (3D motion). The suspension was centrifuged, and the supernatant was loaded onto a Ni 2+ -NTA-agarose column equilibrated with 20 mM Tris-HCl, 300 mM NaCl, pH 7.5. The elution of the NBs was performed with 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, and pH 7.5. The collected fractions were analyzed by SDS-PAGE and pooled. After that, an extensive dialysis step was performed (4 °C, in a 20 mM Tris-HCl, 300 mM NaCl, pH 7.5). Protein purity was > 95%, as evaluated in SDS-PAGE. No aggregation was observed, as inferred from size exclusion chromatography (SEC) analysis. Pure NB was stored at − 70 °C until use. When osmotic shock was performed, protein preparations of higher purity were obtained compared with lysis by sonication. Hydrodynamic Behavior of the NB:FXN Protein Complexes SEC-FPLC was performed using a Superose-6 column (GE Healthcare). Protein concentration was 6–10 µM, a volume of 100µL was typically injected, and the running buffer was 20 mM Tris-HCl, 100 mM NaCl, at pH 7.4. The experiment was conducted at room temperature (∼25°C) at a 0.5 mL/min flow rate. A JASCO HPLC instrument was used. It had an automatic injector, a quaternary pump, and a UV-VIS UV-2075 (elution was monitored at 280 nm). Thermal Shift Assay Temperature-induced denaturation of NB:FXN complexes using the pathogenic G130V FXN variant was monitored by the change in the Sypro Orange dye fluorescence using protein at a 5.0 µM concentration in 50 mM sodium phosphate buffer, pH 7.0. Samples of FXN G130V alone, wild-type FXN alone or NB alone, and samples without any protein were also included as controls. The dye was used at 2 × (as suggested by Thermo Fisher Scientific). The temperature slope was 1 °C min − 1 (from 20 to 90°C). Excitation and emission ranges were 470–500 and 540–700 nm, respectively. The fluorescence signal was quenched in the aqueous environment but became unquenched when the probe was bound to the apolar residues upon unfolding. Experiments by triplicate were carried out in a Step One Real-Time-PCR instrument (Applied Biosystems, CA, U.S.A.). NB Titration by NMR and the NB Interaction Sites on the FXN Surface 15 N labeled FXN was prepared as before [ 48 ]. Samples for NMR experiments contained 0.1 mM 15 N-labelled protein in a buffer supplemented with 5% D 2 O. NMR experiments were performed at 22° C in a Bruker 600 MHz Avance III spectrometer equipped with a TXI probe. The NMR data were processed with NMRPipe [ 49 ] and analyzed using NMRViewJ [ 50 ]. 1 H- 15 N HSQC experiments were performed to follow the interaction by chemical shift perturbations. The 15 N labeled FXN was titrated with each NB at molar ratios of 0:1, 0:33, 0.66, and 1:1 (NB: 15 N-FXN). The experiment was performed in a 25 mM Tris-HC, 150 mM NaCl, pH 7.4 buffer. Biolayer Interferometry Experiments The experiments were carried out using a BLItz instrument (Sartorius). For this experiment, a recombinant FXN H177C variant (> 98% pure) was labeled with a bifunctional biotin-maleimide probe of long arm Cat# SP-1501-12 (Vector). Variant FXN H177C-biotin was purified by G25 (separated from the free probe), and extensive dialysis was performed and stored at -70 °C. The sensors were hydrated in TBS buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.4) for these experiments and supplemented with 0.5 mg/mL BSA (TBS-BSA) for 10 minutes. Subsequently, FXN-biotin binding to the streptavidin sensor surface was carried out. Each experiment consisted of a baseline (100 sec, 250 µL), an association (300 sec, 4 µL), and a dissociation (300 sec, 250 µL). After each experiment, the sensor was regenerated by glycine 10 mM pH 1.0 (5 sec, 300 µL) and TBS-BSA buffer washing. Cell Culture, Treatments, and Materials HEK-293T cells (kindly provided by Dr. Ibanez, INQUIMAE, UBA) and Hela Kyoto cells (kindly provided by Dr. Matias Blaustein, iB 3 , UBA) were grown in high glucose (4.5 g L − 1 glucose) Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Natocor), penicillin/streptomycin (100 units mL − 1 and 100 µg mL − 1 respectively, Thermo Fisher Scientific) and 110 mg L − 1 of sodium pyruvate (Thermo Fisher Scientific) in a 37°C humidified incubator containing 5% CO 2 . Polyethylenimine (PEI, PolyAR, UBA) was used for transfection. Briefly, cells were plated (2 ×10 6 HEK-293T cells per 100 mm plate, 5×10 4 HeLa Kyoto cells per well in 12 well plate) and grown for 24 h before transfection. pCMV_MTS_NB_4A7, pCMV_MTS_NB_6B1, pCMV_MTS_NB_16C10, and pCMV_MTS_NB_28F6 vectors encoding each NB (optimized for mammalian cells expression) preceded by the mitochondrial transit signal (MTS) from the citrate synthase enzyme for the mitochondrial matrix localization, were transfected according to the PEI manufacturer’s instructions. For the immunofluorescence assays, p-ds-Red2-mito_vector (kindly provided by Dr. Roxana Gorojod, IQUIBICEN, UBA) was co-transfected with the MTS_NB in the same conditions. After transfection, cells were grown for 48 h. SDS-PAGE and Western Blotting Assay Protein lysates were prepared using RIPA low salt buffer (20 mM Tris-HCl, 150 mM NaCl, 0,1% between 20, 2 mM EDTA, pH 7.40) and complete protease inhibitor solution (Thermo Fisher). Protein concentration was determined using a Bradford reagent (Thermo Fisher). Protein samples (either purified or complete lysates) were boiled in sample buffer (4% SDS, 20% glycerol, 120 mM Tris, pH 6.8, 0.002% bromophenol blue, 200 mM 2-mercaptoethanol) and subjected to 16% SDS-PAGE. Electrophoresis was carried out at room temperature for 20 min at 90V and 1.5h at 150V. Proteins were stained with Coomassie Brilliant Blue G-250. When Western blotting analyses were performed, proteins were transferred to either a PVDF or a nitrocellulose membrane (0.2 µm, Thermo Fisher and BioRad) for 1 h at 100 V. Membranes were blocked for 1 h at room temperature with 5% skimmed milk in 0.05% tween TBS buffer. Blocked membranes were incubated overnight at 4°C with either an anti-human FXN mAb (abcam, ab 110328), an anti-His6 mAb (MA-125, Thermo Fisher), or an anti-GAPDH mAb (SC-47724). Afterward, HRP-conjugated anti-mouse was incubated for 1h at room temperature and visualized by enhanced chemiluminescence (Clarity Substrate, Biorad) using Amersham Imager 680. Aconitase and Succinate Dehydrogenase Activity after NB Transfection Briefly, HEK-293T cells were plated (2×10 6 cells per 100 mm plate) and grown for 24 h before transfection (using PEI) with pCMV_MTS_NB_4A7, pCMV_MTS_NB_6B1, pCMV_MTS_NB_16C10, and pCMV_MTS_NB_28F6, according to the PEI manufacturer’s instructions (an empty vector control was included). After 48 h, protein lysates were prepared using either PBS buffer or aconitase reaction buffer and complete protease inhibitor (Thermo Fisher). Protein concentration was determined using a Bradford reagent (Thermo Fisher). Succinate dehydrogenase activity was measured using the Succinate Dehydrogenase Activity Assay Kit (Colorimetric) (Abcam; ab228560). Aconitase activity was measured using the Aconitase Enzyme Kit (Abcam, ab109712). All assays were performed following the manufacturer’s instructions. Mitochondrial Oxygen Consumption Rate Measurements after NB Transfection The mitochondrial OCR and ECAR were monitored in vivo in real-time using a Seahorse XFp analyzer (XFp, Agilent). For this, HEK-293T cells were plated (2 ×10 5 cells in a 6-well plate) and grown for 24 h before transfection (using PEI) with pCMV_MTS_NB_4A7, pCMV_MTS_NB_6B1, pCMV_MTS_NB_16C10, and pCMV_MTS_NB_28F6, according to the PEI manufacturer’s instructions (an empty vector was included as control condition). After 24 h of transfection, 2.5 × 10 4 cells/well were plated on pre-coated (with PEI, Sigma) Seahorse XFp Cell Culture Miniplates (Agilent) and maintained in culture conditions for 24 h, when bioenergetics was assessed. Seahorse assays were performed on confluent plates (criterion: cells covering 99.90 ± 0.05% of the plate surface, quantified over a grid of 25 × 25 µm). This criterion was confirmed by quantification of Höescht-positive nuclei at the end of the Seahorse assays (data not shown). On the day of the assay, the culture medium was aspirated and replaced by XF Base Medium (Agilent) supplemented with 25 mM D-glucose, 1 mM sodium pyruvate, and 2mM l-glutamine, pH 7.4. Cells were incubated with this medium for 1 h at 37°C in a non-CO 2 incubator, and then the microplate was loaded into the Seahorse XFp Analyzer (Agilent) following the manufacturer’s instructions. Cells were titrated with 0.125 − 2.0 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to render the maximum oxygen consumption rate (OCR), and these concentrations were used for experiments. The OCR was determined at the beginning of the assay (basal OCR) and after the sequential addition of 1.0 µM oligomycin (Oligo), 1.0 µM FCCP, and 0.5 µM rotenone plus antimycin A (Rot/AA). Three basal rates and three response rates (after adding a compound) were measured, and the average of these rates was used for data analysis. Respiratory parameters were obtained as follows: Basal respiration is the baseline OCR. Respiration driving proton leak is the OCR after the addition of 1µM Oligo. Respiration driving ATP synthesis is the Basal respiration minus Respiration driving proton leak. Maximum respiration is the OCR after the addition of 1 µM FCCP. Spare respiratory capacity (SRC) is the Maximum respiration minus the Basal respiration. Values were expressed as a percentage of the OCR corresponding to the last baseline rate (100%). Data was normalized to the cell number at the end of the assay. Co-Immunoprecipitation Assay HEK-293T cell lysates (500 µg) were incubated overnight at 4°C under agitation with 4 µg of a 6×-His tag antibody (MA 1-135, Thermo Fisher). Binding to protein G magnetic beads (Biorad) was performed for 1 h at 4 ºC (previously, beads were washed with the same RIPA buffer). Three washes were performed after binding and then eluted with 25 µL of a 1× sample buffer (for SDS-PAGE) containing 8% 2-mercaptoethanol. After that, the co-immunoprecipitated proteins were analyzed by western blotting. LC-MS analysis For mass spectrometry analysis, the SDS-PAGE fragments were treated according to LaBaer and coworkers [ 51 ]. A volume of 4 µL of each sample was injected. Peptide separations were performed on a nano HPLC Ultimate3000 (Thermo Scientific) using a nano column EASY-Spray ES901 (15 cm × 50 µm ID, PepMap RSLC C18). The mobile phase flow rate was 300 nL/min using 0.1% formic acid in water (solvent A) and 0.1% formic acid and 100% acetonitrile (solvent B). The gradient profile was set as follows: 4–30% solvent B for 64 min, 30–80% solvent B for 7 min and 80% solvent B for 1 min. MS analysis was performed using a Q-Exactive HF mass spectrometer (Thermo Scientific). 1,9 kV of liquid junction voltage and 250°C of capillary temperature were used for ionization. The entire scan method employed a m/z 375–2000 mass selection, an Orbitrap resolution of 120,000 (at m/z 200), a target automatic gain control (AGC) value of 1e6, and a maximum injection time of 100 msec. After the survey scan, the 15 most intense precursor ions were selected for MS/MS fragmentation. Fragmentation was performed with a normalized collision energy of 28 eV, and MS/MS scans were acquired with a dynamic first mass, AGC target was 5e5, resolution of 30000 (at m/z 200), isolation window of 1.4 m/z units, and maximum IT was 55 ms. Charge state screening was enabled to reject unassigned, singly charged, and equal or more than six protonated ions. A dynamic exclusion time of 25 sec was used to discriminate against previously selected ions. Mass Spectrometry Data Analysis Using standardized workflows, MS data were analyzed with Proteome Discoverer (versión 2.4.1.15). Mass spectra *.raw files were searched against a database from Homo sapiens (UP000005640) and NB-16c10 sequence. Precursor and fragment mass tolerance were set to 10 ppm and 0.02 Da, respectively, allowing two missed cleavages. The following modifications were set: -Max. Equal modifications per peptide: 3. Max Dynamic Modifications per peptide: 4. The dynamic modifications included in the analysis were (i) oxidation (+ 15.995 Da), (ii) N-terminal modification, acetylation (+ 42.011 Da), (iii) N-terminal modification, Met-loss (-131.040 Da), (iv) N-terminal modification Met-loss plus acetylation (-89.030 Da); the static modification included was the carbamidomethylation (+ 57.021 Da). Immunofluorescence Assays For fluorescent reporter assays, cells were plated either into 12-well plates with precision cover glasses or 8-well (Nunc® Lab-Tek® II) coverglass imaging plates. After 48h transfection, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS, and permeabilized for 5 min with 0.2% Triton X-100 in the same buffer. Blocking was performed for 1 h in 1% BSA solution. After incubation for 1 h with the primary (anti-VHH, rabbit polyclonal antibody) and the secondary antibody (Alexa 488 labeled anti-rabbit antibody, Thermo Fisher) in blocking solution for 1h, cells were extensively washed with PBS, and nuclei were stained with Hoescht dye. Images were captured on an Olympus FV-1000 fluorescence microscope with a 60 × oil immersion objective and a Coolsnap HQ2 CCD camera (Photometrics). Assembly of the NB:FXN Complexes For structural purposes, FXN:NB_16C10, FXN:NB_4A7 and FXN:NB_6B1 complexes were assembled by incubation and then copurified by using the His-tag present in the nanobodies. Briefly, the complexes were copurified by HisTrap FF column using an imidazole gradient from 20 mM to 500 mM in 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 1 mM DTT. The complexes were eluted at 130 mM of imidazole. The eluted fractions containing the desired complexes were identified using SDS-PAGE, and concentrations were determined by NanoDrop. All the proteins were collected and dialyzed for imidazole removal using a Spectra/PorTM 1RC dialysis membrane (of 6–8 kDa MWCO). The resulting protein solutions were concentrated on an Amicon Ultra-4 Centrifugal Filter (MWCO 10kDa; Millipore Sigma, Burlington, MA). The final concentration for FXN:NB_16C10, FXN:NB_4A7, and FXN:NB_6B1 was 7.5mg/mL, 3.5mg/mL, and 11mg/mL, respectively. NB:FXN Crystallization Experiments Initial crystallization screening using the copurified complexes was performed at 298K using the sitting-drop vapor-diffusion method with an available collection of commercial conditions. The drops consisted of 0.2µL of protein solution (7.5 mg/mL, 3.5 mg/mL or 11mg/mL of protein complex in a buffer containing 30 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM DTT) and 0.2 µL well solution, and were equilibrated against 60uL of well solution. The extensive screening rendered crystals in several conditions and their corresponding diffraction quality was checked on the beamline BL13-XALOC at the ALBA Synchrotron (Barcelona, Spain). Best diffracting crystals were subsequently scaled up and optimized, yielding crystals grown in 15% PEG 10K, 0.1M ammonium acetate and 0.1 M Bis-Tris pH 5.5 (FXN:NB_16C10); 0.2M Na thiocyanate pH 6.9, 20% PEG 3350 (FXN:_NB4A7); and 0.1 M HEPES pH 7.5, 0.8 M potassium sodium tartrate (FXN:NB_6B1). X-ray Data Collection and Processing All data were collected from frozen crystals at 100 K with the PILATUS 6M detector at beamline XALOC (ALBA Synchrotron, Barcelona, Spain). Data processing and scaling were accomplished using XDS [ 52 ], POINTLESS, and AIMLESS [ 53 ] as implemented in autoPROC [ 54 ]. Statistics for the crystallographic data and structure solution are summarized in Table S1 . Crystal Structure Solution, Model Building and Refinement The NB:FXN structures were solved by the molecular-replacement method, as implemented in the program PHASER [ 55 , 56 ] using ab initio models given by AlphaFold [ 57 , 58 ] as search models. Then, its initial models were subjected to iterative cycles of model building and refinement with Coot [ 59 ] and REFMAC [ 60 ], respectively. Final refinement cycles were performed with PHENIX [ 61 ], yielding the refinement and data collection statistics summarized in Table S1 . Figures were generated using PyMOL [ 62 ] and ChimeraX [ 63 ]. The final refinement parameters are summarized in Table S1 . Small Angle X-ray Scattering (SAXS) SAXS experiments were performed at the beamline B21 of the Diamond Light Source [ 64 ]. Samples of 45 µL of all the complexes at different concentrations were loaded onto an SRT-C SEC-300 (Sepax) column equilibrated in buffer (20 mM Tris-HCl pH 8.0 and 150 mM NaCl) and connected to an Agilent 1200 HPLC system at 18°C. The continuously eluting samples were exposed for 3s in 10s acquisition blocks using an X-ray wavelength of 1 Å and a sample-to-detector (Eiger 4M) distance of 3.7 m. The data covered a momentum transfer range of 0.0032 < q < 0.34 Å−1. The frames recorded immediately before the sample elution were subtracted from the protein scattering profiles. The Scåtter software package ( www.bioisis.net ) was used to analyze data, buffer-subtraction, scaling, merging, and checking possible radiation damage of the samples. The Rg value was calculated with the Guinier approximation, assuming that at very small angles q < 1.3/Rg. The particle distance distribution, Dmax, was calculated from the scattering pattern with GNOM, and shape estimation was carried out with DAMMIF/DAMMIN; all these programs are included in the ATSAS package [ 65 , 66 ]. The protein molecular mass was estimated with GNOM. Interactively generated PDB-based homology models were made using the program COOT by manually adjusting the X-ray structures obtained in this work, into the envelope given by SAXS until a good correlation between the real-space scattering profile calculated for the homology model matched the experimental scattering data. This was computed with the program FoXS [ 67 ]. RESULTS Sequence Analysis and Structure Predictions of the NB/FXN Complexes After the phage display selection using native mature FXN as the target, and a preliminary L-Cys-desulfurase activity screening, a total of 30 NBs specific for human FXN were initially sequenced. We found a substantial sequence diversity for the complementary determining regions (CDRs) CDR1, CDR2, and CDR3 (Figs. 1 D and 2 ), yielding 16 different sequences. Eleven NBs were chosen for protein expression in E. coli WK6 strain, purification, and in vitro studies. Four NBs (NB_4A7, NB_6B1, NB_16C10, and NB_28F6, Table S2 ) and the corresponding NB:FXN complexes were deeply studied in this work. Alpha-Fold 3 [ 57 , 58 , 68 ] were used to infer structures of NB:FXN complexes from their amino acid sequences. The results suggested many different types of complexes NB:FXN might be formed ( Figure S1 ). More than one different NB:FXN complex type was usually predicted for each NB sequence. Furthermore, predictions indicated that some NBs might display an inhibitory effect because the binding region comprises a surface of FXN involved in the supercomplex architecture (e.g., an ISCU2 or NFS1 interacting surface). On the other hand, some NBs might interact with FXN surface areas that are not involved in inter-subunit interactions. In those cases, NBs may not affect the activation exerted by FXN. However, NBs may fix a particular conformation, altering the internal motions of the FXN and, therefore, modulating the FXN function. NB Expression in E. coli WK6 NBs were purified by NTA-Ni 2+ , yielding 95% pure protein (5–15 mg/L). In all cases, monomeric conformations were obtained, as judged by SEC-FPLC (see below). For NB_28F6, protein degradation was observed when the NB was stored at 4°C. Thus, we decided to preserve the NBs at -70°C. Apart from NB_28F6, which exhibited proteolysis, the masses of the purified NBs were in the 2 Da range of the expected masses, as deduced from the amino acid sequences, considering the predicted processing site after NB export to the periplasmic space (the processing of the signal peptide results in an N-term QVQLQ, Fig. 2 and Table S2 ). NB Effect on Supercomplex Activity in vitro After nanobody purification, we investigated the effect of NBs addition to the enzymatic reaction on the L-Cys desulfurase catalysis (Figs. 3A and S2) . The NBs NB_4A7 and NB_16C10 exhibited a low-middle degree of inhibition, while NB_6B1 and NB_28F6 showed higher inhibition of the in vitro L-Cys-desulfurase activity. It is worthy of note that the complex (NFS1/ACP-ISD11/ISCU) 2 shows lower activity than the core, (NFS1/ACP-ISD11) 2 . In fact, one can infer ISCU interaction with (NFS1/ACP-ISD11) 2 from the observed decrease in the desulfurase activity [ 69 ]. Higher NB concentrations of NB_6B1 and NB_28F6 produced the highest decrease in the activity of the supercomplex like the one detected in the virtual absence of FXN, which represents a drop in the value corresponding to (NFS1/ACP-ISD11/ISCU) 2 . On the other hand, the inhibitory effect of higher concentrations of NB_16C10 was lower than the observed for NB_6B1 and NB_28F6, suggesting that NB_16C10 affects the conformation or the topology of the supercomplex but would not interfere with the FXN-supercomplex interaction in these experimental conditions. The activity of the supercomplex in the presence of the higher level of NB_4A7 was the highest observed among the NBs studied in vitro in this condition (Fig. 3B-E) . In Vitro Characterization of the NB:FXN Interaction To evaluate the in vitro NB:FXN complex formation, we first studied the interaction by size-exclusion chromatography (SEC). Purified NBs, FXN, or the mix of both proteins (injection after a 10-minute incubation of FXN and NB at room temperature) were loaded in an analytical SEC system. As judged by the SEC profiles, many of the selected NBs against FXN formed stable complexes (Figs. 4 and S3 ), and their profiles suggested a slow binding equilibrium. Besides this, the SEC profile corresponding to NB_15C5 was compatible with the absence of complex formation exhibiting two peaks consistent with free FXN and free NB ( Figure S3C ). The binding between NBs and FXN was studied employing biolayer interferometry (BLI). To evaluate the interaction, a biotin-labeled FXN variant (H177C, Figure S4 ) was immobilized in a streptavidin sensor at 10 µg mL − 1 . The selected NBs exhibited high affinity with equilibrium dissociation constant K D in the nanomolar range 1–33 nM, and association and dissociation kinetic rate coefficients k a and k d in the range 10 5 M − 1 s − 1 and 10 − 4 M, respectively (Fig. 4 and Table S3 ). Identification of the NB Binding Site on the FXN Surface The binding sites of this subset of NBs were studied with a resolution at the level of the amino acid residues by NMR, analyzing the chemical shift perturbations (CSP). FXN was produced in E. coli using the 15 N source 15 NH 4 Cl and purified for these experiments. The target was then titrated with each of the NBs, and 1 H- 15 N-HSQC NMR bidimensional spectra were acquired. Then, CSP and changes in the cross-peak intensities were analyzed (Figs. 5 and S5-S8 ). CSP results indicated that NBs NB_4A7, NB_6B1, and NB_16C10 bind to a similar site on the FXN surface (residues involved are shown in Table S4) . Instead, NB_28F6 presents a different binding mode, as indicated by the residues involved in the interaction. As judged by the discontinuous pattern of CSP observed for almost all the cross peaks involved, the results suggested, in all cases, a binding mechanism characterized by a slow dissociation equilibrium. Remarkably, some residues from the FXN core exhibited significant CSP, including aromatic side chains Phe110, Tyr123, and Phe127 and the aliphatic residue Leu113. This suggests that the interaction between NB and FXN may adjust some conformational details of the FXN structure. In the case of NB_28F6, some residues far from the suggested interaction site also exhibited high CSP ( Table S4 ). Among them, Tyr143, Ile145, and Trp155, the latter involved in the assembly site of the supercomplex. Modulation of the FXN Conformational Stability by NB Interaction To test whether NB interactions could stabilize the FXN conformation, we conducted in vitro temperature-induced unfolding experiments. Protein denaturation was monitored by the fluorescence of the Sypro-orange probe. Briefly, when the dye interacts with the unfolded state of proteins, the quantum yield increases, exhibiting an increase in the fluorescence intensity. We used FXN G130V as a probe for highly unstable Friedreich’s Ataxia (FRDA) variants [ 70 ] for these experiments. This variant is unstable in vitro and in vivo , giving rise to very low FXN concentrations in the mitochondrial matrix in vivo [ 71 ]. Noteworthy, the NBs were able to significantly stabilize the G130V FXN variant as judged by a significant shift (≥ 17 °C) in the observed Tm values (e.g., Tm values are 51.8 ± 0.3 and 70.1 ± 0.1°C, for the G130V variant and NB_4A7: G130V_FXN complex, respectively, Figs. 6A and E ). The Tm value corresponding to the complex is even higher than that observed for the wild-type FXN (Tm = 65.9±0.3°C). Similar results were obtained for the rest of the NBs (Fig. 6 ) , suggesting that they might stabilize the native conformation of FXN in the cellular environment. On the other hand, Sypro-orange dye did not show significant interaction with the nanobodies used in this assay in this range of temperature (blue lines in Fig. 6 A-D ). Three-dimensional Structure of NB:FXN Complexes To get high-resolution information on the binding mode of the NBs studied in this work, NB_4A7, NB_6B1, and NB16C10 in complex with wild-type FXN (90–210 variant) were crystallized, and their structures solved (Fig. 7). The three crystallographic complexes were solved at atomic resolutions of 1.25 for FXN: NB_4A7, 1.48 for FXN: NB_6B1 complex, and 2.0 Å for FXN: NB_16C10 complex. The quality of the electron density map was excellent ( Figure S9 ), allowing clear modeling of both proteins and fully identifying the residues involved in protein-protein interaction. FXN residues involved in the interaction are listed in Table S5 . The structure of human FXN in our complexes presents a nearly identical structure to that reported previously (PDB ID: 1EKG) [ 72 ], with RMSD values ranging from 0.21–0.24 Å (for superimposition of all Cα atoms). FXN presents two parallel α-helices supported by a platform provided by a five-stranded, antiparallel β-sheet. The NBs adopt the typical immunoglobulin fold, with ten β-strands forming two β-sheets connected by loops and a conserved disulfide bond between Cys22 and Cys95 (Fig. 2 A ). The NB:FXN crystal structures confirmed the interaction surface and binding regions observed by NMR experiments (CSP results, Fig. 5 and Table S4 ). In all cases, the two proteins in the complex are arranged in a T fashion mode with FXN orienting the acidic ridge (the loop linking α1 and β1, residues Glu114-Glu122), the loop β2–β3 (residues 135–139) and the tip of helix α1, against the central part of the NB β-sandwich. Specifically, the NBs interact with frataxin through the loop β3–β4 (residues Arg38-His46, using Fig. 2A numbering), the β4-strand (residues Leu47-Arg50), and the loop β5–β6 (residues Asp61-Lys64); NB_4A7 and NB_16C10 also interact through Asn58 from β4 (Fig. 7). Remarkably, the CDR regions of the NBs are not directly involved in binding to FXN. Some relevant electrostatic interactions are observed in the NB:FXN complex (e.g., Glu121 FXN -Arg38 NB , Lys135 FXN -Asp61 NB , Asp139 FXN -Lys64 NB , Phe120 FXN -Arg50 NB ) (Fig. 7), involving a molecular contact surface area of 581.7 Å 2 . Structural comparison of FXN alone and in complex with NB ( Figure S10 ) reveals that major changes upon NB interaction are in Loop 1, L1, connecting α1–β1, and Loop 2, L2, connecting β2–β3 (Fig. 7 and Figure S10 ). While some variations are also observed in β5–β6 turn and the loop connecting β6 and α2, depending on the NB. Overall, a similar pattern of interactions is conserved for the interaction of FXN and NB_4A7, NB_6B1, and NB_16C10, where loops L1 and L2 and a punctual interaction through α2 are pivotal for protein-protein recognition. D122Y and G137V mutations in FXN (two pathogenic FRDA variants found in patients) occur in residues located on the interface of the NB:FXN complexes, ( Figure S11 ). Whether these NBs can interact and stabilize a highly unstable variant as G137V [ 33 ] should be investigated. Whereas this pathogenic FXN variant activated the supercomplex and did not exhibit structural alterations, it shows a significant decrease in its conformational stability that is correlated to the very low FXN concentration found in people living with FRDA, considered the main feature of the physiopathology of this variant. SAXS studies on FXN:16C10 and FXN:28F6 complexes in solution The complex FXN:NB_28F6 did not produce crystals in our hands; thus, we decided to perform Small Angle X-ray Scattering (SAXS, Table S6 ) studies to check if the interaction would follow the same mode observed for NBs NB_16C10, NB_6B1 and NB_4A7. The complex FXN:NB_16C10 was also measured for comparison with our crystal structures. In both cases, we observed a good superimposition between the experimental and theoretical scattering curves (Fig. 8 A ). As expected, the FXN:NB_16C10 complex, the low-resolution envelope in solution, fits with the crystal structure of the complex in which the two proteins are oriented in a T-fashion mode (Fig. 8 D ). However, the FXN:28F6 complex shows a new disposition in which the two proteins pack in a parallel fashion with the b-sheets from both proteins configured as a single long b-sheet (Fig. 8 C ). The model observed in the solution for the FXN:NB_28F6 complex is compatible with the predicted arrangement provided by AlfaFold3 ( Figure S1 D ). As discussed below, this different arrangement for the NB_28F6 can explain the observed differences in the behavior in our cellular experiments. How the NB:FXN Complex Docks onto the Supercomplex Structure Whether the selected NBs might block the interaction of FXN with the rest of the supercomplex depends on the specific structure of the NB:FXN complex and how it fits/perturbs into the supercomplex structure throughout the catalytic cycle. To gain structural information on that hypothesis, we combined information provided by X-ray diffraction experiments concerning NB:FXN complexes (from this work) with previous structural data regarding the supercomplex (NFS1/ACP-ISD11/ISCU/FXN) 2 provided by cryo-electron microscopy (PDB ID: 6NZU [ 5 ]). The NB:FXN structures (Fig. 9A) were docked onto the supercomplex by the superimposition of the FXN subunit. In the type I NB:FXN binding (NBs NB_4A7, NB_6B1, and NB_16C10, Fig. 9B ), only slight clashes between the NB and the rest of the subunits of the supercomplex were predicted. The contacts that the NB may establish through its β3–β4 connector with one of the NFS1 subunits might lead to the inhibitory effects observed in vitro . In the case of NB_16C10, residues involved in the clashes may be Pro41 NB -Asn245 NFS1 , Gly42 NB -Arg271 NFS1 , Lys43 NB -Glu120 NFS1 , Arg45 NB -Arg273 NFS1 , and His46 NB -Arg273 NFS1 (using the numbering shown in Fig. 2A for the NB and the numbering used in PDB ID: 6NZU for NFS1). Whether the plasticity of the protein chains might properly accommodate this stretch could depend on the amino acid sequence of the NB β3–β4 connector (Fig. 9 B ), thus determining the rigidity/flexibility behavior and, ultimately, the inhibitory power of these NBs that bind using the same site. Given that NB_4A7 and NB_16C10 have an extra Gly in that stretch, we expect more flexibility than in the case of NB_6B1, in which an Asn residue occupies the place of the Gly44. The edition by reengineering β3–β4 connectors will be a key for obtaining better FXN binders. Remarkably, when the supercomplex formed by NFS1, ACP-ISD11, ISCU2, and FXN subunits was loaded in SEC-FPLC system, in the presence of NB_4A7, the elution of the complete assembly shifted to a lower elution time (from 37.65 to 37.28 min, black arrow 2 to 3, absence or presence of NB_4A7) (Fig. 9 C ). By contrast, when FXN was absent, the presence of NB_4A7 did not modify the elution time of the subcomplex (NFS1/ACP-ISD11/ISCU) (Fig. 9 C , elution time 38.4 min, black arrow 1 dark and light green, with or without NB_4A7), a fact indicative of the NB specificity for FXN. More importantly, these results suggest that NB_4A7 can bind FXN in the context of the supercomplex. Additionally, it was evident that a fraction of the NB bound to the free FXN shifted its elution time to lower values, indicating the stabilization of the complex NB:FXN (elution time shifted from 44.4 to 43.3 min in the absence or presence of NB_4A7, respectively). It is worthy of note that under these experimental conditions, the interaction of FXN with NFS1 is not so strong; in fact, other research groups reported a dissociation constant for wild-type FXN of K D ~3±1 µM [ 5 ]. Thus, we expected a high fraction of unbound FXN (∼55% unbound, ∼45% bound [ 73 ]). Moreover, considering the dilution due to the SEC column volume, the unbound FXN fraction may be higher (a half dilution would result in ∼72% unbound and ∼28% bound). We also carried out an interferometry experiment to investigate whether supercomplex and NB_4A7 can simultaneously bind to FXN (Fig. 9 D ). In this experiment, streptavidin sensors were sensitized using the biotin-labeled FXN H177S variant, ( Figure S4 ), and after that, sensors were incubated with solution containing both NB and the subcomplex (NFS1/ACP-ISD11/ISCU2) 2 , in the presence of a 3:1 excess of ISCU2: NFS1/ACP-ISD11/ISCU2) 2 , L-Cys and PLP to increase the affinity between ISCU2 and the core complex (NFS1/ACP-ISD11) 2 [ 11 ]. Our results showed that the signal corresponding to the complete mix of proteins is even higher than the observed for the NB_4A7 alone, suggesting that the interaction of FXN with the NB_4A7 does not impede the supercomplex formation. Additionally, the K D measured for FXN: (NFS1/ACP-ISD11/ISCU2) 2 . interaction was ∼0.6 µM, a value in the range of the dissociation constant previously measured under similar conditions (0.2 µM) [ 11 ]. On the other hand, when a similar experiment was carried out using NB_28F6 (Fig. 9 E ), which binds to the helix 1, the signal observed for the subcomplex (NFS1/ACP-ISD11/ISCU2) 2 and NIAI + NB_28F6 was similar to that observed for NB_28F6. Suggesting that this NB inhibits the binding of the subcomplex to FXN, in agreement with structural results (SAXS and NMR). Expression of NBs in the Mitochondria of Human Cell lines To evaluate the effects of NB:FXN interaction on the cellular metabolism, we first studied whether the NBs could be successfully imported to the mitochondrial matrix when expressed in human cell lines. We used transfected HeLa Kyoto cells to evaluate this by immunofluorescence. We prepared vectors that included the NB sequence in frame with the citrate synthase mitochondrial transit sequence (MTS) for mitochondrial matrix localization. The cells were co-transfected with the Discosoma red fluorescent protein (dsRed Mito), which also included an MTS (cytochrome c oxidase subunit VIII). The transfected cells were analyzed after 48 hours post-transfection. As expected, when NBs were detected using an anti-VHH polyclonal serum, which recognizes the NBs invariable regions (followed by an anti-rabbit secondary antibody labeled with Alexa 488), the NB signal co-localized with that obtained with dsRed-Mito, strongly suggesting a mitochondrial localization of the NBs. This behavior was observed for the four nanobodies studied in this work (Fig. 1 0 ). Importantly, the expression of NBs NB_4A7, NB_6B1 and NB_16C10 did not alter the viability of the cells ( Figure S12 ). Therefore, we demonstrated that the NBs are localized in the mitochondria and that their expression did not significantly affect the cell viability. In turn, even though the expression of NB_28F6 was very low, it was possible to evaluate its subcellular localization, and the analysis suggests that it is in mitochondria. On the other hand, the viability analysis of cells transfected with NB_28F6 should be examined cautiously because of the lower expression levels and the possibility that the effects were not comparable to the rest of the NBs. NB Expression and Interaction between FXN and NB in HEK-293T Cells. Characterization of Fe-S clusters related to mitochondrial bioenergetics Co-immunoprecipitation was used to investigate whether the NBs can interact with FXN in a cellular environment. The NB candidate, NB_16C10 in this case, was expressed in HEK-293T cells, and its interaction with endogenous FXN was explored. Noteworthy, endogenous FXN was co-immunoprecipitated with NB_16C10 using an anti-Histag antibody, which indicated that a stable NB:FXN complex was formed in the cells (Fig. 1 1 ). Additionally, samples corresponding to the co-immunoprecipitation were investigated by mass spectrometry. The analysis of the peptides corresponding to the recovered proteins indicated a strong interaction between the NB_16C10 and FXN ( Table S7 ). Furthermore, peptides corresponding to citrate synthase suggested that the NB precursor containing the MTS of this mitochondrial enzyme can interact with FXN in the cellular environment. This result points to future investigations concerning whether the NB and FXN could interact in the cytosol or, conversely, the precursor of the NB might interact with FXN in the mitochondrial matrix. Remarkably, no proteins other than FXN, NB, and those recovered when cell samples were transfected with the empty vector were consistently retrieved, suggesting that the method used for cellular lysis may perturb the stability of other complexes involving protein-protein interactions of lower affinities than that of NB:FXN ( K D for FXN in the nanomolar range, Table S3 ). The effect of NB expression on FXN expression was studied. The densitometric analysis of western blot membranes (three independent experiments) showed similar levels of FXN among the cells transfected with an empty vector ( Figure S13A ). Besides this, the expression of NB_4A7, NB_6B1, and NB_16C10 in HEK-293T cells was substantial ( Figure S13B ). However, the expression of NB_28F6 was significantly lower ( Figure S13B ) than the others (~ 90% lower, as evidenced by western blotting). Transfected HEK-293T cells were evaluated to identify possible adverse effects of the NB expression on [Fe-S] cluster-dependent enzymatic activities from aconitase (ACO) and succinate dehydrogenase (SDH). The expression of NBs showed only slight modulations of ACO and SDH activities (Figs. 1 2 A and B) . Only the expression of the NB_16C10 led to a slight decrease of the SDH enzyme in HEK-293T cells (Fig. 1 2 B) . More experiments will be carried out to evaluate whether this is a direct effect on the enzyme through the alteration of Fe-S cluster assembly activity or, on the other hand, the modulation of SDH is the consequence of more complex processes involving transcription or/and translation, or even post-translational modifications. To gain a more global picture of the effect of the FXN_ NB expression in these cells, we measured the oxygen consumption rate (OCR) 48h after transfection (Figs. 1 2 C-F ), which is indicative of the oxidative phosphorylation (OXPHOS) efficiency. Our results suggested that the OCR is not altered when NB_6B1 or 16C10 are expressed. These NBs exhibited similar extents in the increase of the OCR after FCCP treatment, revealing a similar maximal respiratory capacity (Fig. 1 2G ), as non-transfected cells. On the other hand, NB_4A7 exhibited a slight decrease in the basal respiratory capacity, possibly compatible with the slight decrease of SDH (Complex II, not significant) activity. However, the observed metabolic changes seem subtle, given the considerable mitochondrial protein expression of NB_4A7, 6B1, and 16C10. In turn, when the HEK-293T cells were transfected with a vector encoding NB_28F6, even though the expression of this NB was significantly lower than that of the other NBs ( Figure S13B ), a decrease in basal and maximal respiration was observed (Fig. 1 2F ), indicative of decreased mitochondrial metabolism. DISCUSSION In this work, we characterized new molecular tools to explore if it is possible to modulate FXN stability and function through its interaction with foreign tutor proteins. With this aim, twenty NBs specific to FXN were selected by phage display. We found that NBs can increase FXN conformational stability. Binding was in the nanomolar range of 1–33 nM, suggesting a strong interaction, a key feature for function modulation purposes. SEC, NMR, and BLI results also suggested a slow dissociation equilibrium. In addition, in vitro L-Cys-desulfurase activity could be modulated by the NBs; while 1:1 molar ratio does not affect L-Cys desulfurase activity for some NBs (NB_4A7, 5A8, 29F7, and 83G4, Figs. 3 and S2 ), an increase to 1:5 ratio was inhibitory. As at 1:1 ratio, ∼83% of the FXN is in complex with NB_4A7 ( K D =33.0 nM), whereas ∼17% is free (calculated using the ligand binding simulation tool [ 73 ]), the inhibition observed at 5:1 indicates a more complex interaction of the NB with the rest of the proteins of the system, suggesting unspecific binding under these conditions. The relevance of this fact will be studied further. On the other hand, binding of NB_4A7 to FXN did not inhibit the interaction of FXN with the supercomplex (Fig. 9), in agreement with the preservation of the ∼85% of Cys desulfurase activity observed in vitro for 1:1 ratio (Fig. 3). Different experimental techniques (NMR, X-ray crystallography, and SAXS) enabled us to characterize the interaction between the FXN and the NBs. Two distinct types of binding, which we have designated as Type I binding (as we find for NBs NB_16C10, NB_6B1, and NB_4A7), and Type II binding (as found for NB_28F6) were found (Figs. 5, 7, and 8 ). The complexes with a Type I binding have two key polar interactions with the FXN residues of the loop L1 of FXN, specifically, the residues Glu121, Pro117, and Thr119, involving a molecular contact surface area of 581.7 Å 2 . Residue Glu121 plays an essential role in the protein interaction of Type I by making a salt bridge interaction with the residue Arg38 and polar interactions with Ser62 of the NBs. Pro117 and Thr119 also play an important role by establishing H-bonds with residues His46 and Leu47 of β5 from the NBs. Even though, Asn52 from CDR2 makes an H-bond with backbone Gly138 from FXN, and Val98, Pro99, Pro100, from CDR3 are at Van der Waals distance of FXN, in Type I interaction mode, the recognition is not made using the three segments CDR1, CDR2, and CDR3. However, this is common for NBs that use a far vaster diversity of structural stretch combinations to bind antigens [ 74 ]. To study the effect of NBs in vivo , human cell lines were transfected with vectors encoding four different NBs. Three presented a considerable expression level; instead, NB_28F6 exhibited significantly lower expression. The four transfected NBs were found in the mitochondria of Hela Kyoto cells. Remarkably, the expression of the NBs in HEK-293T and HeLa Kyoto cells did not alter FXN expression or cell viability. Considering the degree of modulation of L-Cys desulfurase in vitro , the higher expression level of NBs in the cells, and the plausible broader spectrum of functions that FXN might exert inside the cells, including direct interactions with iron, aconitase [ 75 , 76 ], superoxide dismutase [ 77 ] or Complex I from the respiratory chain [ 78 ], we reasoned that the expression of the NBs could affect the Fe-S cluster-dependent enzymatic activities and energetics. However, the expression of NB_6B1 and NB_4A7 did not have effects over Fe-S dependent ACO and SDH activities, and the expression of NB_16C10 neither had effects over ACO but exhibited a slight inhibition over SDH (Fig. 1 2 ). This last fact could be compatible with the absence of in-cell inhibition of mitochondrial L-Cys desulfurase NFS1 supercomplex in HEK-293T cells overexpressing NBs. Moreover, in a more global picture of the mitochondrial metabolism, we demonstrated that the expression of NB_4A7, NB_6B1 and NB_16C10 in HEK-293T did not alter the mitochondrial respiration 48 h after transfection, suggesting that the mitochondrial global metabolism is not significantly perturbed when these NBs are expressed, imported into the mitochondria and eventually interacting with FXN (as judged by co-immunoprecipitation assay). Remarkably, NB_28F6 yielded a similar profile of ACO and SDH activities. However, under the same conditions, NB_28F6 showed very poor expression levels (∼5 times lower in average, Figure S13 ). Thus, we could not evaluate its effect on metabolism with confidence. The observed decrease of the OCR (even with a considerably lower protein expression level than the other NBs) suggests that NB_28F6 expression affects mitochondrial OCR through FXN intervention. In particular, the binding surface of FXN involved in the interaction with NB_28F6 could explain the inhibitory effects observed in vitro and in vivo (Figs. 3 and 13 ). As evidenced by our NMR results, it involves a larger portion of helix α1 in FXN, the acidic ridge of FXN and NB_28F6 binding resulted in the inhibition of the FXN-supercomplex interaction (Fig. 9). Unfortunately, the crystal structure of the FXN: NB_28F6 complex could not be obtained. We consider that some heterogeneity in the sample due to partial proteolysis of this NB (mass spectrometry results) might impede the crystallization process. These different results between the in vitro and in-cell effects can be understood considering that the in vitro measured activity involves only a partial reaction, the first steps of Cys desulfurase reaction, employing DTT as a non-physiological reducing agent. In contrast, in the cell, the Fe-S cluster formation to sustain Fe-S dependent enzymes involves several steps, including the persulfide transfer to ISCU2, the electron delivery (reduction) by FDX2, the iron-sulfur cluster assembly on ISCU2, and the cluster transferring to target subunits and enzymes. The dynamic behavior of the L-Cys NFS1 desulfurase supercomplex was further characterized last year by including the electron donor for cluster assembly Ferredoxin 2 (FDX2) [ 20 ]. Recently, it was determined that this protein has a multifaceted binding mode (two steps involving the C-terminal partially folded stretch of FDX2) to the supercomplex [ 79 ]. Moreover, it was demonstrated that FDX2 binds to the same supercomplex region as FXN. This indicates that precise exchange dynamics are essential for accurate function. Changes in FXN or FDX2 affinity by the supercomplex could significantly impact the kinetics of the catalytic cycle. The interaction of FXN with the NB, making subtle clashes of the NB and NFS1 structures, might have unexpected effects on function, suggesting that the reaction studied in vitro , the L-Cys desulfurization in the presence of DTT, may show a different profile compared to the complete reaction occurring in the cell, involving the Fe-S cluster formation, which depends on FDX2/FXN dynamics. This work suggests the possibility of rescuing pathogenic FXN variants characterized by lower conformational stability and an increased tendency to be degraded compared to wild-type FXN and opens the door to a general strategy for intervening in human mitochondrial biochemistry. Abbreviations ACP acyl carrier protein CD circular dichroism CTR C-terminal region Fe-S iron-sulfur FA Friedreich’s Ataxia FXN frataxin DTT dithiothreitol HPLC high-performance liquid chromatography‎ FPLC Fast protein liquid chromatography ISCU iron-sulfur cluster assembly enzyme ISD11 NFS1 interacting protein NFS1 mitochondrial L-cysteine desulfurase enzyme NMR nuclear magnetic resonance PAGE polyacrylamide gel electrophoresis PDB Protein Data Bank SDS sodium dodecyl sulfate SEC size exclusion chromatography. Declarations Acknowledgments : This work was supported by Universidad de Buenos Aires UBACyT20020190100338BA, CONICET, and Friedreich´s Ataxia Research Alliance (FARA, grant 2023-2025). Author contributions: M.F.P. and J.S. performed experiments, made the analysis of the experiments and structures, and wrote the paper. N.B. F. made co-immunoprecipitations, western blots, and analysis of the results A. G., R. M., and J. A. H. 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Supplementary Files NCSuppInfo26022025.docx Supplementary Information Nanobodies as Novel Tools to Modulate Human Frataxin Stability and Function PDBXValidationReportsNCOMMS2515806.zip Maps and Models and PDB Validation Reports Cite Share Download PDF Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Biociencias, Biotecnología y Biología Traslacional (iB3), Universidad de Buenos Aires, Buenos Aires, Argentina","correspondingAuthor":false,"prefix":"","firstName":"Augusto","middleName":"","lastName":"García","suffix":""},{"id":426769123,"identity":"7a8c8cd1-88fe-4d8a-ae61-4cf1b7ed7de8","order_by":10,"name":"Hernán Gentili","email":"","orcid":"","institution":"Instituto de Biociencias, Biotecnología y Biología Traslacional (iB3)","correspondingAuthor":false,"prefix":"","firstName":"Hernán","middleName":"","lastName":"Gentili","suffix":""},{"id":426769124,"identity":"6d34623c-b619-4029-9ede-8f5dc2bcfd69","order_by":11,"name":"Naira Rodríguez","email":"","orcid":"","institution":"Instituto de Biociencias, Biotecnología y Biología Traslacional (iB3), Universidad de Buenos Aires, Buenos Aires, Argentina","correspondingAuthor":false,"prefix":"","firstName":"Naira","middleName":"","lastName":"Rodríguez","suffix":""},{"id":426769125,"identity":"95e29761-4980-4eb5-a949-5966fd3aec3a","order_by":12,"name":"Martín Aran","email":"","orcid":"","institution":"Fundación Instituto Leloir, IIBBA‐CONICET.","correspondingAuthor":false,"prefix":"","firstName":"Martín","middleName":"","lastName":"Aran","suffix":""},{"id":426769126,"identity":"a0c947bb-b07a-4c15-9a3b-739d93d51bd3","order_by":13,"name":"Viviana Parreño","email":"","orcid":"","institution":"Incuinta, Instituto Nacional de Tecnología Agropecuaria (INTA).","correspondingAuthor":false,"prefix":"","firstName":"Viviana","middleName":"","lastName":"Parreño","suffix":""},{"id":426769127,"identity":"86246c72-8492-446c-b207-38e4429f5d35","order_by":14,"name":"Marina Bok","email":"","orcid":"","institution":"Incuinta, Instituto Nacional de Tecnología Agropecuaria (INTA).","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"Bok","suffix":""},{"id":426769128,"identity":"76b551f1-f772-493e-bcae-d3cf64ea8574","order_by":15,"name":"Juan Hermoso","email":"","orcid":"https://orcid.org/0000-0002-1862-8950","institution":"Rocasolano Physical-Chemistry Institute. CSIC","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Hermoso","suffix":""},{"id":426769129,"identity":"12aee861-07f0-4a57-9859-509d3173a7ee","order_by":16,"name":"Lorena Ibañez","email":"","orcid":"https://orcid.org/0000-0002-9393-2004","institution":"Instituto de Ciencia y Tecnología Dr. César Milstein CONICET","correspondingAuthor":false,"prefix":"","firstName":"Lorena","middleName":"","lastName":"Ibañez","suffix":""},{"id":426769112,"identity":"341ea7a2-c9eb-4ec7-b5eb-0c6e00d59a76","order_by":17,"name":"Javier Santos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBACxgYGhgMIbgUQMzM3EKmFDUScAWlhxK8FAUBaGNtgxuABzO1nDA983FMnby7ffPgz77zaaP52oJYfFdtwO6wnx+DgjGeHDXe2saVJ8247njvjMGMDY8+Z23j8kpZwmOfAAcYNx3jMmHO3HcttAGphZmzDo6X/WcLhPwfq7IFajD/nzjmWO5+glhnJBw4zHGBOBGoxkM5tqMndQFjL4wMHew4cTt5wLC1N+s+xA7kbgVoO4vOLYX9i84cfB+psNxw+fPjjjJq63HnnDx988KMCj5YGVP5hMHkAp3ogkEfj1+FTPApGwSgYBSMUAAAGomQd0c5EgQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1140-8234","institution":"Instituto de Biociencias, Biotecnología y Biología Traslacional (iB3), Universidad de Buenos Aires, Buenos Aires, Argentina","correspondingAuthor":true,"prefix":"","firstName":"Javier","middleName":"","lastName":"Santos","suffix":""}],"badges":[],"createdAt":"2025-02-27 15:10:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6122246/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6122246/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-09458-x","type":"published","date":"2026-01-03T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78326807,"identity":"739d561c-3020-4c12-aa61-1a8372752850","added_by":"auto","created_at":"2025-03-12 06:33:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":709945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial Fe-S cluster Biosynthesis. \u003c/strong\u003e(A and B)\u003cstrong\u003e \u003c/strong\u003eTwo views of the supercomplex are involved. Cys desulfurase NFS1, ACP, ISD11, ISCU, and FXN are shown in a surface representation. Only part of the supercomplex [5] is shown in (C), the Cys381 of the NFS1 enzyme, ISCU2, and FXN are shown—additionally, the PLP cofactor, FXN Trp155, and ISCU assembly site. The distance between PLP and Cys381 is ~15Å, and between Cys381 and the ISCU2 assembly site is ~15Å. ISD11 and ACP subunits were omitted for clarity. (D)\u003cstrong\u003e \u003c/strong\u003eA scheme of a nanobody (V\u003csub\u003eHH\u003c/sub\u003e domain) structure showing the three complementarity determining regions: CDR1, CDR2, and CDR3. The N- and C- extremes are indicated.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/95cff80e763bb73a6ff88103.png"},{"id":78326808,"identity":"6a2cfb23-6a97-4537-86b9-7713cd70e97a","added_by":"auto","created_at":"2025-03-12 06:33:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":939747,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe selected Nanobody Sequences. \u0026nbsp;\u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eMultiple sequence alignment (MSA) of the 16 different selected sequences. The CDR regions are shown: CDR1 (precursor: 49-55, mature: 26-33), CDR2 (precursor: 72-83, mature: 52-59), CD3 (precursor: 119-139, mature: 97-115). (B) The signal peptide for periplasm localization (MKYLLPTAAAGLLLLAAQPAMA), the HA-tag (YPYDVPDY), and the His-tag, which are included in all NBs, are shown in the context of the consensus NB sequence (CDRs are highlighted as in A). The figure was prepared using the SnapGene program, and the MSA was carried out using the Muscle algorithm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/6ce24d6cde0c7d62911b0b49.png"},{"id":78326851,"identity":"8bd39d47-638b-432d-a359-343b1c9ed13c","added_by":"auto","created_at":"2025-03-12 06:33:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":215467,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of Cys Desulfurase Activity by NB:FXN Interaction.\u003c/strong\u003e (A) The effect of the addition of NBs on the Cys-desulfurase supercomplex activity for NB_4A7, NB_6B1, NB_16C10, and NB_28F6). “NIAIF” refers to the reaction, including NFS1/ACP-ISD11/ISCU/FXN, in the absence of NB. NIA refers to a sample without ISCU2, FXN and NB, and corresponds to the activity of the core (NFS/ACP-ISD11)\u003csub\u003e2\u003c/sub\u003e, whereas NIAI corresponds to the core plus ISCU2, but without FXN. “-Pro” corresponds to a reaction in the absence of proteins. We also studied 5:1 molar ratio. (B) NB_4A7, (C) NB_6B1, (D) NB_16C10, and (E) NB_28F6.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/fe04239f262895bb5387b16b.png"},{"id":78326812,"identity":"6b1948d4-f068-442b-8ab4-70019fb56b98","added_by":"auto","created_at":"2025-03-12 06:33:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":355661,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNB:FXN Interaction Characterized by SEC-FPLC and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBLI\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe interaction between NBs and FXN was followed by SEC for (A) NB_4A7, (B) NB_6B1, (C) NB_16C10, and (D) NB_28F6. The SEC profiles corresponding to the isolated FXN (black and gray, having three different concentrations at 2, 3 and 6mM) or NBs (color lines). Dashed lines show the profiles corresponding to a 1:1 NB:FXN mixture. The lower elution volume for the mixture indicates the formation of a stable complex. The interaction followed by BLI. Biotin-labeled FXN variant (H177C) was titrated with each NB. Representative binding assay corresponding to (E) NB_4A7, (F) NB_6B1, (G) NB_16C10, and (H) NB_28F6 are shown. The experiment was performed in a 25 mM Tris-HC, 150 mM NaCl, pH 7.4 buffer, and 0.5 mg mL\u003csup\u003e-1\u003c/sup\u003e BSA was added in all solutions to avoid unspecific binding of FXN and NB to the sensor and tube surfaces. NB solutions typically contain from 0 to 150 nM NB concentration. The global fitting is in black lines.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/868ec125a1f440d6fa8af2df.png"},{"id":78326809,"identity":"d0dc7780-3caf-4495-9694-2b1683a65080","added_by":"auto","created_at":"2025-03-12 06:33:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1001388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNB Interaction Sites on FXN surface Studied by NMR. \u003c/strong\u003eFXN was titrated with each NB at molar ratios of 0:1, 0:33, 0.66 and 1:1 (NB:\u003csup\u003e15\u003c/sup\u003eN-FXN), shown in blue, cyan, magenta and light green, respectively; (A) and (B): NB_4A7, (D) and (E): NB6B1, (G) and (H): NB_16C10, (J) and (K): NB_28F6 correspond to selected regions from the FXN spectra illustrating some of the residues exhibiting different patterns of CSP after incubation with the NB for each complex. (C): NB_4A7, (F): NB_6B1, (I): NB_16C10, and (L): NB_28F6, the residues exhibiting the highest CSP were mapped on the FXN structure (using a van der Waals style of representation for the residues involved and colored by element). The complete spectra are shown in \u003cstrong\u003eFigures S5-S8\u003c/strong\u003e. The experiment was performed in a 25 mM Tris-HC, 150 mM NaCl, pH 7.4 buffer. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC experiments were performed. A list of the residues involved is given as Supplementary Information (\u003cstrong\u003eTable S4\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/a9f7e0c81e1ee861e49aa30c.png"},{"id":78326811,"identity":"b917b2a2-6ed5-4455-8584-dab2044c59e4","added_by":"auto","created_at":"2025-03-12 06:33:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":326246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemperature-Induced Unfolding of NB:FXN G130V Complexes. \u003c/strong\u003eExperiments were carried out on a multi-well plate, and Sypro-orange fluorescence was monitored. Wild type and G130V FXN variants without NB were included as controls in black (Tm=65.9±0.3°C) and gray (Tm=51.8±0.3 °C), respectively. A, B, and C correspond to NB_4A7, NB_6B1, and NB_16C10, respectively. Tm values for the complexes are 70.1±0.1, 68.7±0.1, and 69.9±0.1 °C for NB_4A7: G130V, NB_6B1: G130V, NB_16C10: G130V, and 66.3 ± 0.1°C NB_28F6:G130V complex, respectively). (D) Tm values for FXN and G130V variants and the complexes FXN G130V:NB. (E) Ribbon model of FXN (PDB ID:1ekg, Gly130 is in gray).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/5b8a789b2285e5af5b35383d.png"},{"id":78326826,"identity":"10888105-351a-4751-9a93-83d7066cfc6e","added_by":"auto","created_at":"2025-03-12 06:33:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":662887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrystal Structures of FXN:NB_16C10, FXN:NB_6B1, and FXN:NB_4A7 complexes. (A\u003c/strong\u003e) Cartoon\u003cstrong\u003e \u003c/strong\u003erepresentation of the FXN:NB_16C10 complex. Boxed areas show the most important interactions between the NB_16C10 and FXN. Relevant residues are depicted as capped sticks and labeled. Polar interactions are represented as dotted lines. (\u003cstrong\u003eB)\u003c/strong\u003e Cartoon\u003cstrong\u003e \u003c/strong\u003erepresentation of the FXN:NB_6B1 complex. Boxed areas show the most important interactions between the NB_6B1 and Frataxin. (\u003cstrong\u003eC)\u003c/strong\u003e Cartoon\u003cstrong\u003e \u003c/strong\u003erepresentation of the FXN:NB_4A7 complex. Boxed areas show the most important interactions between the NB_4A7 and FXN. X-ray crystallographic data collection and refinement statistics of NB:FXN complexes are in \u003cstrong\u003eTable S1.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/e537a32cc04b26abf628c349.png"},{"id":78329576,"identity":"0dd4fbf4-d787-45fc-99dd-7f4bfdb16131","added_by":"auto","created_at":"2025-03-12 06:57:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":372268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis by SAXS of Complexes FXN:NB_28F6 and FXN:NB_16C10. (A) \u003c/strong\u003eFit of the experimental scattering curve (green and pink dots) and theoretical scattering (green and pink lines) computed for the model of FXN:NB_28F6 and FXN:NB_16C10. (\u003cstrong\u003eB)\u003c/strong\u003e Plot showing the normalized pair-distance distribution function P(r) for the complexes reflecting the distance distribution.\u003cstrong\u003e (C) \u003c/strong\u003eSuperimposition on the ab initio-determined SAXS envelope (pale grey) for FXN:NB_28F6 complex with the AlphaFold3 predicted model. (\u003cstrong\u003eD)\u003c/strong\u003e Superimposition on the ab initio-determined SAXS envelope (pale grey) for FXN:NB_16C10 with its crystal structure-based model.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/67b35f4634c0b51eaf79b6fc.png"},{"id":78326835,"identity":"a128a73a-ad8c-4303-885c-3132e0765fec","added_by":"auto","created_at":"2025-03-12 06:33:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":234325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNB:FXN Complex Mapped on the Supercomplex.\u003c/strong\u003e (A) The X-ray structure of complex FXN:NB_16C10 was superimposed to the cryo-EM structure of the supercomplex (PDB ID: 6NZU). Gray surfaces represent NFS1subunits and ISCU2. (B) Residues from NB_16C10 involved in the possible clash with NFS1subunit are shown. (\u003cstrong\u003eC)\u003c/strong\u003e SEC-FPLC profile of the supercomplex (NFS1/ACP-ISD11/ISCU2/FXN)\u003csub\u003e2\u003c/sub\u003e in the presence of NB_4A7 (blue) or in the absence of the NB_4A7 (light blue). The SEC profile corresponding to the subcomplex (NFS1/ACP-ISD11/ISCU2)\u003csub\u003e2\u003c/sub\u003e in the presence of the NB_4A7 (dark green, NIA/I/NB) or in the absence (light green, NIA/I). FXN alone or NB_4A7 alone were also loaded (black and orange, respectively). Buffer or buffer plus PLP profiles are shown as controls (dashed black and gray lines, respectively). (D) Interaction between biotin-FXN and NB_4A7 (orange dashed line), the subcomplex (NFS1/ACP-ISD11/ISCU2)\u003csub\u003e2\u003c/sub\u003e (NIAI, orange dotted line), or biotin-FXN with NB_4A7 and subcomplex (NFS1/ACP-ISD11/ISCU2)\u003csub\u003e2\u003c/sub\u003e (NIAI+NB_4A7, orange full line) followed by Interferometry. (E) Interaction between biotin-FXN and NB_28F6 (orange dashed line), the subcomplex (NFS1/ACP-ISD11/ISCU2)\u003csub\u003e 2\u003c/sub\u003e (NIAI, orange dotted line), or biotin-FXN with NB_28F6 and (NFS1/ACP-ISD11/ISCU2)\u003csub\u003e 2\u003c/sub\u003e (NIAI+NB_28F6, orange full line) by Interferometry. Buffer was 25mM Tris-HCl, 150mM NaCl, 1mM DTT, 1mM Cys, 10mM PLP, pH 8.0. (NFS1/ACP-ISD) concentration was 1mM, and ISCU was 3mM.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/ada8b7d51f4d8398600ed9d1.png"},{"id":78326849,"identity":"0f0a86e9-23fb-4e07-b0a4-4fb24dedc067","added_by":"auto","created_at":"2025-03-12 06:33:12","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1144347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular Localization of NBs in HeLa Kyoto Cells. \u003c/strong\u003eThe expression of NBs was performed for 48h. Transfections were carried out with polyethyleneimine (PEI) and 1 mg of total DNA (e.g., 500ng pTwist_NB + 500 ng Ds_Red_mito plasmid). Anti VHH rabbit polyclonal antibody (1/800 in 1% BSA in 1´PBS), antirabbit Alexa 488 (Thermo Fisher). Hoechst was used for nuclei staining. The instrument was Olympus FV1000. Excitation was performed at 405, 488, and 543 nm. The scale bar corresponds, in all cases, to 10.0 mm.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/9475b810d169eea72bd359ba.png"},{"id":78329892,"identity":"37b1e7db-aa4d-4445-90e0-75f086d714dd","added_by":"auto","created_at":"2025-03-12 07:05:14","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":175436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation of NB:FXN. \u003c/strong\u003eHEK-293T cells were transfected with an empty vector or a vector encoding the NB_16C10 sequence for protein expression. IP: His indicates the lanes where the co-immunoprecipitation (was performed using an anti-His tag antibody). FXN was detected by an anti-FXN monoclonal antibody, whereas NB was detected using the anti-His antibody. The GADPH enzyme was evaluated to measure the protein mass present in the input of the co-IP experiments. The symbols + and – indicate the presence or absence of the corresponding vectors for cell transfection (empty or encoding NB_16C10).\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/2df4c3ec07f651ca5db1ef5e.png"},{"id":78329571,"identity":"5da3204a-167a-4d58-9927-7474106985df","added_by":"auto","created_at":"2025-03-12 06:57:11","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":218675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioenergetic Profiles, Nanobody Expression, Enzymatic Activities, and Oxygen Consumption Rates in HEK-293T Cells. \u003c/strong\u003e(A) Aconitase and (B) Succinate dehydrogenase activities (SDH and ACO, respectively) were quantified 48h after transfection.\u003cstrong\u003e \u003c/strong\u003eOCR profiles are shown for cells transfect with (C) NB_4A7, (D) NB_6B1, (E) NB_16C10, and (F) NB_28F6. (G) The OCR parameters. After basal O\u003csub\u003e2\u003c/sub\u003e consumption recording, oligomycin (an ATP synthase inhibitor) is added, and the coupling to the ATP synthesis is measured.\u0026nbsp; Then, the addition of FCCP (a protonophore that uncouples oxygen consumption from ATP synthesis) allows us to measure the maximum respiratory capacity to use OXPHOS. This also enables the measurement of the spare respiratory capacity (SRC) as the difference between maximal and basal OCR. Finally, rotenone and antimycin A (Complex I and Complex III inhibitors, respectively) are added to block the electron transport chain.\u003c/p\u003e","description":"","filename":"image13.png","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/e729ca9888746c0428d98980.png"},{"id":102094483,"identity":"a1aa7293-c7d4-40ae-b62f-4a88b54a07ca","added_by":"auto","created_at":"2026-02-07 08:07:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8241091,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/c78b510f-b86b-42b7-adbc-d8863aebec01.pdf"},{"id":78328078,"identity":"79167d0a-f9c2-4fcc-8419-d2c4ed61747b","added_by":"auto","created_at":"2025-03-12 06:41:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5025320,"visible":true,"origin":"","legend":"Supplementary Information Nanobodies as Novel Tools to Modulate Human Frataxin Stability and Function","description":"","filename":"NCSuppInfo26022025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/fbf595d83420c8be1495c3c9.docx"},{"id":78328072,"identity":"0718f862-e2f3-4efb-bd4b-795fa14d8730","added_by":"auto","created_at":"2025-03-12 06:41:11","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6469522,"visible":true,"origin":"","legend":"Maps and Models and PDB Validation Reports","description":"","filename":"PDBXValidationReportsNCOMMS2515806.zip","url":"https://assets-eu.researchsquare.com/files/rs-6122246/v1/969570b975e873f404eaa21d.zip"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nNational patent application pending (in Argentina): INPI_P5476AR00 (CONICET-UBA).","formattedTitle":"Nanobodies as Novel Tools to Modulate Human Frataxin Stability and Function","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe study at the molecular level of iron-sulfur cluster biosynthesis has recently increased the comprehension of some of the relationships between structure and function concerning the mitochondrial supercomplex involved in this pathway. The Fe-S clusters are at the core of many essential biological activities. Mitochondrial enzymatic functions such as NADH dehydrogenase, aconitase, succinate dehydrogenase, and complex III from the respiratory chain, lipoic acid synthetase, and processes such as nuclear DNA repair [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], the chemical modifications of transfer RNAs that affect base recognition between codon and anticodon [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and the cytoplasmic Fe-S cluster assembly by the cytosolic iron-sulfur cluster assembly (CIA) system require the mitochondrial cysteine desulfurase supercomplex [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This supercomplex consists of several proteins, all of them encoded by nuclear DNA (Fig.\u0026nbsp;1) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. NFS1 cysteine desulfurase is one of the essential enzymes. It is translated as a 50 kDa precursor, imported into the mitochondrial matrix, and processed to a mature dimeric form (89 kDa) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This enzyme is a dynamic hub that collects many other proteins on its surface. The L-Cys desulfurization reaction depends on pyridoxal phosphate (PLP), and although it is multifaceted, it can be summarized in a series of key stages [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]: a) formation of the outer Schiff base with the L-Cys substrate; b) formation of the persulfide-enzyme intermediate; c) release of L-Ala and transfer of the persulfide to a Cys in a mobile loop of the enzyme (Cys381 in the case of human NFS1); and d) the subsequent transfer of the -SH group to the iron-sulfur cluster assembly enzyme (ISCU2). In subsequent reactions, the persulfide groups will be reduced and combined with iron to form the Fe-S clusters. ISCU2 is an essential scaffold protein that supports the assembly of the [2Fe-2S] cluster at its active site, which is composed of two cysteines (Cys95 and Cys138), an aspartic acid (Asp71), and one histidine (His137) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Crystallographic data show that the ISCU2 active site is in close contact with the Cys-loop of the NFS1, where Cys381 is located [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt was demonstrated that the L-cysteine increases the binding efficiency of human ISCU2 to NFS1 nine-fold (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e = 0.2 \u0026micro;M with cysteine and 1.7 \u0026micro;M without [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Downstream, trafficking proteins and targeting factors, including a dedicated system of chaperones and co-chaperones, transfer the Fe-S cluster to acceptor proteins (e.g., enzymes) [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother essential protein that forms the supercomplex is frataxin (FXN), which is the kinetic activator of the NFS1 and accelerates the persulfide transfer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. FXN is imported into the mitochondrial matrix as a 210-residue precursor and processed in two steps; the first produces an intermediate form (residues 41 to 210) and after that, the second stage produces the mature FXN form 81\u0026ndash;210 (14.2 kDa) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Current structural (Cryo-EM) and functional studies suggest that FXN simultaneously interacts with ISCU2 and with both subunits of the NFS1 dimer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, it was shown that the natural source of electrons is provided by the ferredoxin system involving NADPH, FdxR, and FDX2 [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Since FXN binds iron through its acidic region involving alpha helix 1, loop 1, and beta-strand 1, with a moderate \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], it is possible that this protein could also participate in the reaction delivering the Fe(II) metal ions to ISCU2, a process necessary for the biosynthesis of the clusters. Current experimental evidence indicates that FXN binds to iron in the context of the protein complex and that FXN could release the metal ion by transferring it to ISCU2 in the presence of L-Cys (the substrate) and the natural reducing agent FDX2 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, how the iron transfer to ISCU occurs remains unclear.\u003c/p\u003e \u003cp\u003eIt was shown that NFS1 is stabilized in its active dimeric form by a small protein called ISD11 (10.5 kDa), which also prevents NFS1 aggregation. ISD11 is an essential protein in eukaryotes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and it is completely absent in Bacteria and Archaea [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, the alterations in the expression or structural dynamics of the proteins that conform to the supercomplex decrease the functionality of the pathway. Notably, decreased expression or function of ISCU2 results in the severe disease ISCU myopathy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A pathogenic ISD11 variant (R68L) has been associated with the development of a mitochondrial genetic disorder, COXPD19. This mutation resulted in the reduction of the affinity between ISD11 and NFS1, increasing the tendency of NFS1 to aggregate and the accumulation of reactive oxygen species, leading to mitochondrial dysfunction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Besides, mutation R72Q of NFS1 was described as the cause of the deficit in NFS1 function, which results in the infantile mitochondrial complex II/III deficiency, an autosomal recessive mitochondrial disease that is characterized by lactic acidemia, respiratory chain complex II and III deficiency, and abnormal mitochondria [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In particular, the decrease of FXN expression results in Friedreich's ataxia (FA), an inherited neurodegenerative rare disease affecting 1 in 50,000 people [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this context, one of our main research goals is to select specific small tutor proteins that may provide conformational stability to pathogenic, highly unstable variants of FXN while preserving key interactions with other proteins of the mitochondrial supercomplex. We have named this strategy quaternary addition [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, we selected affi_224, a synthetic FXN binder, employing the Ribosome Display strategy and archaeal Sac7D protein as the scaffold (Affitins) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We showed that Affi_224, fused to a human mitochondrial targeting sequence, was successfully expressed in HEK-293T cell lines and interacted with FXN, as judged by co-immunoprecipitation. Moreover, when the wild-type FXN is replaced by the G130V variant, the presence of affi_224 increases the activity of the supercomplex \u003cem\u003ein vitro\u003c/em\u003e. However, the latter did not occur when the wild-type FXN was replaced by the highly unstable W173G variant. In this sense, other \u003cem\u003ein vitro\u003c/em\u003e experiments showed that Affi_224 did not form a stable complex with FXN. Furthermore, affi_224 is an unstable protein \u003cem\u003eper se\u003c/em\u003e, and it tends to form dimers. These features might preclude its use in further studies.\u003c/p\u003e \u003cp\u003eWith this background, we explored other scaffolds for developing FXN tutors to investigate the stabilization strategy and chose the camelid-derived nanobody scaffold (V\u003csub\u003eHH\u003c/sub\u003e or NBs). Highly specific NBs can be obtained by phage display strategy from an RNA library of camelid antibody sequences prepared after a convenient immunization of the camelid with the target protein. Camelid heavy chain antibodies are formed by two heavy chains, each one containing a variable domain (V\u003csub\u003eHH\u003c/sub\u003e), which includes three complementarity-determining regions (CDR1, CDR2, and CDR3, the latter longer than the others) (Fig.\u0026nbsp;1\u003cb\u003eD\u003c/b\u003e). NBs are small (15kDa) and conformationally stable proteins [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] that can be easily and cheaply produced compared to conventional monoclonal antibodies. NBs can be expressed in \u003cem\u003eE. coli\u003c/em\u003e. In addition, their small size may allow better tissue penetration [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Even though NBs usually display low immunogenicity, they can be humanized to reduce this characteristic and, therefore, be suitable for therapeutic applications in humans [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. NBs can be expressed by mammalian cells as \"intrabodies\" while retaining their conformational stability. In particular, an advantage of NBs is that they can be delivered by gene therapy as single genes; these proteins are currently being used to explore treatments for various neurodegenerative disorders and cancer disease, and some of them are in clinical trials [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this paper, we selected more than twenty FXN-specific NBs. The interaction between FXN and four of them was deeply investigated \u003cem\u003ein vitro\u003c/em\u003e and in a cellular environment.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDNA Sequencing, Protein Concentration, and Mass Spectrometry Analysis\u003c/h2\u003e \u003cp\u003eIdentity was verified by DNA sequencing using the Macrogen facility. Absorption spectra were obtained with a JASCO V-730 BIO spectrophotometer (Tokyo, Japan). Protein concentration was determined using the extinction coefficient obtained from the amino acid sequence using the ProtParam tool from ExPASy. Protein purity was estimated by SDS-PAGE analysis, and the analysis of the intact masses was performed by mass spectrometry in the National Laboratory of Research and Services in Peptides and Proteins using an LCQ DUO ESI ion trap (Thermo Finnigan) or QExactive Orbitrap (Thermo Scientific) spectrometers.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHPLC Analysis of Protein Samples\u003c/h3\u003e\n\u003cp\u003eReverse phase HPLC analysis was performed using a JASCO system equipped with an autoinjector, UV detector, and a thermostatic oven at 25 \u0026deg;C. Gradients from 0 to 100% acetonitrile were done, and 0.05% TFA (\u003cem\u003ev/v\u003c/em\u003e) were added to the solvents and samples. The columns used were analytical C18 (Higgins Analytical, Inc. U.S.A.), and the flow was 1.0 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Proteins were monitored at 220 nm. Before injection, all samples were centrifuged.\u003c/p\u003e\n\u003ch3\u003eExpression and Purification of the Human Supercomplex Core (NFS1/ACP-ISD11)\u003c/h3\u003e\n\u003cp\u003eThe DNA sequences of the human mature form of the human cysteine desulfurase NFS1 enzyme (NFS1Δ55), the ISD11, and the human mitochondrial acyl carrier protein (ACP, the mature form) were optimized for protein expression in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) by BIO BASIC Inc (Markham ON, Canada). NFS1Δ55 and ISD11 were cloned in a pETDuet-1 plasmid, whereas ACP was cloned in a pACYCDuet-1 for co-expression. NFS1 amino acid sequence included the RSGHHHHHH tag in the N-terminal for purification and antibody recognition. Protein expression was induced by 1mM IPTG when the bacterial culture reached OD\u003csub\u003e600nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0. Co-expression was carried out overnight at 20\u0026deg;C (at 250 rpm). The purification of the complex (NFS1/ACP-ISD11)\u003csub\u003e2\u003c/sub\u003e was performed from the soluble fraction of \u003cem\u003eE. coli\u003c/em\u003e BL21 DE3 cultures using a Ni\u003csup\u003e2+\u003c/sup\u003e-NTA-agarose column (first step). The protein was eluted with 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0. After that, 2 mM DTT was added and the protein was dialyzed overnight against the same buffer, without imidazole, at 4\u0026deg;C. Afterwards, it was frozen at -70\u0026deg;C. This protocol minimized aggregation and oxidation of the protein. Before each experiment, (NFS1/ACP-ISD11)\u003csub\u003e2\u003c/sub\u003e was thawed in ice and centrifuged. Protein concentration was determined spectroscopically using an absorption coefficient ε\u003csub\u003e280nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;53750 M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eExpression and Purification of ISCU2\u003c/h3\u003e\n\u003cp\u003eThe ISCU2 DNA sequence corresponding to the mature form was optimized for \u003cem\u003eE. coli\u003c/em\u003e overexpression by Explora Biotech (Rome, Italy) and subcloned in a pE22b plasmid, with a C-terminal His6 tag. Protein induction was carried out by adding 1mM IPTG final concentration (3h, 37\u0026deg;C and 250 rpm). ISCU2 was purified using a Ni\u003csup\u003e2+\u003c/sup\u003e-NTA-agarose column equilibrated with 20 mM Tris-HCl, 300 mM NaCl, pH 7.5. The elution was performed with 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 7.5. The collected elution fractions were analyzed by SDS-PAGE and pooled. After that, an extensive dialysis step was performed (4 \u0026deg;C, in a 20 mM Tris-HCl, 300 mM NaCl, pH 7.5, 1 mM DTT). Protein purity was \u0026gt;\u0026thinsp;95%, as evaluated in SDS-PAGE. No aggregation was observed, as inferred from size exclusion chromatography (SEC) analysis. The protein was stored at \u0026minus;\u0026thinsp;70 \u0026deg;C until use. Protein concentration was determined spectroscopically using an absorption coefficient ε\u003csub\u003e280nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11,460 M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (1 mg/mL protein solution represents Abs\u003csub\u003e280nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.70). The zinc content in ISCU samples was evaluated by atomic absorption spectroscopy at the Department of Bromatology, College of Pharmacy and Biochemistry, \u003cem\u003eUniversidad de Buenos Aires\u003c/em\u003e. Zinc concentration in ISCU2 preparations was 0.14: 1 (zinc: ISCU2, molar ratio).\u003c/p\u003e\n\u003ch3\u003eExpression and Purification of FXN\u003c/h3\u003e\n\u003cp\u003eHuman FXN (residues 90\u0026ndash;210) was overexpressed and purified as previously described for the wild-type protein [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Briefly, bacteria cultures (\u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3), 2\u0026ndash;3 L Terrific Broth, pH 7.2) were grown at 37 \u0026deg;C and 280 rpm. Protein expression was induced at DO\u0026thinsp;=\u0026thinsp;0.8-1.0 with 1.0 mM IPTG. After induction (3.5 hours), bacteria were centrifuged (6,000 rpm), and the pellet was stored at \u0026minus;\u0026thinsp;20 \u0026deg;C until cell disruption by sonication (in an ice-water bath). The soluble fraction was separated by centrifugation (10,000 rpm, 30 min). The soluble fraction was incubated with 10 mM EDTA and loaded onto an ion exchange chromatography (DEAE DE52 matrix). The protein was eluted with a 300 mL linear gradient from 0.0 to 1.0 M NaCl (the buffer was 20 mM Tris-HCl, 1 mM EDTA, pH 7.0). Fractions with FXN (identified by SDS-PAGE) were loaded onto a Sephadex G\u0026ndash;100 column (SEC, 93 cm \u0026times; 62.7 cm, equilibrated with 20 mM Tris-HCl, 100 mM NaCl, 1.0 mM EDTA, pH 7.0). FXN concentration was determined spectroscopically using an absorption coefficient ε\u003csub\u003e280nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;26,930 M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (1 mg/mL protein solution represents Abs\u003csub\u003e280nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.00). Purity was \u0026gt;\u0026thinsp;98% as evaluated in SDS-PAGE.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNFS1 Cysteine Desulfurase Activity\u003c/h2\u003e \u003cp\u003eFor enzymatic desulfurization of L-Cys to L-Ala and sulfide by the (NFS1/ACP-ISD11/ISCU/FXN)\u003csub\u003e2\u003c/sub\u003e supercomplex, concentrations of proteins, substrate and the reducing agent DTT were set according to a previous paper by Tsai and Barondeau [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Reactions contained 1.0 \u0026micro;M NFS1/ACP-ISD11, 3.0 \u0026micro;M ISCU, and 1.0 \u0026micro;M FXN, and samples were supplemented with 10 \u0026micro;M PLP, 2.0 mM DTT and 1.0 \u0026micro;M FeSO\u003csub\u003e4\u003c/sub\u003e (final concentrations). In all cases, the reaction buffer was 50 mM Tris-HCl and 200 mM NaCl, pH 8.0, and reactions were started by the addition of 1.0 mM L-Cys (or variable L-Cys concentrations, as we describe below). Samples were incubated at room temperature (25\u0026deg;C) for 30 min. For the reactions including NBs, each NB was preincubated with FXN (10 min), and then, both proteins were added to the reaction mix.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe Methylene Blue Method\u003c/h3\u003e\n\u003cp\u003eSulfide was determined by the methylene blue method [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. For this analysis, H\u003csub\u003e2\u003c/sub\u003eS production was stopped by adding 50 \u0026micro;L of 20 mM \u003cem\u003eN,N\u003c/em\u003e-dimethyl \u003cem\u003ep-\u003c/em\u003ephenylenediamine in 7.2 M HCl and 50 \u0026micro;L of 30 mM FeCl\u003csub\u003e3\u003c/sub\u003e (prepared in 1.2 M HCl). Under these conditions, the production of methylene blue took 20 min. After that, samples were centrifuged for 5 min at 12000 x \u003cem\u003eg\u003c/em\u003e, and the supernatant was separated. Absorbance at 670 nm was measured.\u003c/p\u003e\n\u003ch3\u003eLlama Immunization and Library Construction\u003c/h3\u003e\n\u003cp\u003eA llama located at INTA\u0026acute;s Camelids Experimental Unit was immunized intramuscularly with 150 \u0026micro;g per dose of human FXN 90\u0026ndash;210 recombinant protein on days 0, 14, 28 and 50. Complete Freund\u0026rsquo;s adjuvant was used for the first dose, and incomplete Freund\u0026rsquo;s adjuvant for the following boosts. Antibody responses were monitored by ELISA on serum samples taken before each immunization. Llama inoculation and sample collection were conducted by trained staff, the study was approved by the Animal Care and Use Committee of INTA (CICUAE) under the protocol N\u0026deg; FR6.2-3/2020. Four days after the last boost, 150 mL of anticoagulated blood was used to isolate lymphocytes by Ficoll Paque Plus (GE Life Sciences, 17-1440-02) filled Leucosep tubes (GBO, 227290), total RNA was extracted (RNAeasy Midi, Qiagen 75144) and cDNA was prepared with oligo(dT) primers (First Strand cDNA Synthesis Kit, Roche 04379012001). VH and VHH genes were amplified with CALL001 (5\u0026rsquo;-GTCCTGGCTGCTCTTCTACAAGG-3\u0026rsquo;) and CALL002 (5\u0026rsquo;-GGTACGTGCTGTTGAACTGTTCC-3\u0026rsquo;) primers. A PCR amplicon of 0.7 kb was purified from gel (Wizard\u0026reg; SV Gel and PCR Clean-Up System, Promega A9282) and then used as a template in a nested PCR to specifically amplify the VHH fragments using VHH-BACK-SAPI (5\u0026rsquo;-CTTGGCTCTTCTGTGCAGCTGCAGGAGTCTGGRGGAGG-3\u0026rsquo;) and VHH-FORWARD-SAPI (5\u0026rsquo;-TGATGCTCTTCCGCTGAGGAGACGGTGACCTGGGT-3\u0026rsquo;) primers. A Golden Gate assembly was done to clone the VHH sequences between two SapI sites of the phagemid vector pMECS-GG, following a protocol previously described [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Electro-competent \u003cem\u003eE. coli\u003c/em\u003e TG1 cells (Lucigen 60502-1) were transformed with the purified ligation mixture and plated on a selective agar medium. A library of 1.8\u0026sdot;10\u003csup\u003e9\u003c/sup\u003e individual transformants was obtained [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSelection of FXN Specific NBs\u003c/h2\u003e \u003cp\u003eTo produce recombinant specific NBs, 1 mL of bacteria from the stock library was grown in 2\u0026sdot;TY until OD\u003csub\u003e600nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6, afterward, VCS M13 helper phage (Stratagene, 200251) was used to infect exponentially growing bacteria. The resulting NB phage display library was panned three times on microtiter plates (Maxisorp Nunc) coated overnight at 4\u0026deg;C with 10 \u0026micro;g of each recombinant protein in 100 \u0026micro;L of PBS and 100 \u0026micro;L of PBS for the negative controls. The next day, wells were washed with PBST (PBS\u0026thinsp;+\u0026thinsp;0.05% Tween 20) and blocked with 2% skim milk in PBST. Approximately 1\u0026sdot;10\u003csup\u003e12\u003c/sup\u003e phage particles were preincubated with 10 \u0026micro;L of blocking solution in 100 \u0026micro;L of PBS for 30 min at room temperature by head-over-head rotation, then added on positive and negative wells and incubated for two hours on a vibrating platform (500 rpm.). During the first panning round, wells were washed 10 times with PBST, while wells were washed 20 and 25 times for the second and third rounds, respectively; 5 min incubation on a vibrating platform (300 rpm) was done every five washes. Specific phage particles were eluted with 0.25 mg/mL trypsin solution (Sigma-Aldrich, T1426) for 30 min followed by neutralization with 4 mg/mL AEBSF solution (Carl Roth, 2931.3). A second elution step was done by adding exponentially growing \u003cem\u003eE. coli\u003c/em\u003e TG1 cells to positive and negative wells that were then incubated for 30 min at 37\u0026deg;C. Trypsin-eluted phage particles were amplified by infection of exponentially growing \u003cem\u003eE. coli\u003c/em\u003e TG1 cells and later superinfected with VCS M13 helper phage. Phage particles obtained after both elution strategies were purified using PEG 6,000/NaCl precipitation and used for the next selection round. To obtain specific binders, individual TG1 colonies were screened by ELISA using periplasmic extract. For this, 95 colonies from the positive wells (different panning rounds and elution strategies) and 1 colony from the negative well were inoculated in 1 mL of 2\u0026sdot;TY medium containing 100 \u0026micro;g/mL ampicillin and 0.1% glucose in a deep well plate. NB expression was induced after bacteria incubation for 3 h at 37\u0026deg;C and 200 rpm. with 1 mM IPTG. After 4 h induction, bacterial cultures were centrifuged, the pellets were frozen and thawed twice to disrupt cells and resuspended in 120 \u0026micro;L of PBS. On the other hand, the periplasmic extract (see below, NB purification) was used to determine the specificity of NB binding and to study their functional activity by the Methylene Blue method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eScreening for Antigen Binders\u003c/h2\u003e \u003cp\u003eFor screening experiments, the study of NB binding to FXN was conducted by ELISA. For this, microtiter plates (Maxisorp, Nunc) were coated overnight at 4\u0026deg;C with 200 ng/well of recombinant FXN or an irrelevant protein as negative controls, diluted in PBS. After three washes with PBST, wells were blocked with 3% skim milk in PBST, and 50 \u0026micro;L of the periplasmic extract was added to each well and incubated at room temperature for 2 h. After washing with PBST to remove excess of NB, specific binding was detected with horseradish peroxidase (HRP)-linked anti-HA antibody (Abcam, ab1190) diluted 1:1500. Finally, 50 \u0026micro;L of TMB substrate (3,3', 5,5' tetramethylbenzidine, BD 555214) was added. Absorbance at 450 nm was measured using an ELISA reader (TECAN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNanobody Production and Purification\u003c/h2\u003e \u003cp\u003eA DNA sequence corresponding to the NB, preceded by a signal peptide for export to the periplasm (MKYLLPTAAAGLLLLAAQPAMA), C-terminal hemagglutinin (YPYDVPDY), and His (HHHHHH) tags, was cloned in an expression vector (pMECS), and \u003cem\u003eE. coli\u003c/em\u003e WK6 was transfected with these constructions. Different expression protocols were carried out (temperature was modified from 20 to 37 \u0026deg;C, and expression was performed for 4 h or overnight). Some differences concerning yield and purity were observed for specific NBs. However, we chose to follow the same protocol for all the NBs. Protein induction was carried out by adding 1.0 mM IPTG final concentration (4h, 37\u0026deg;C and 190 rpm). Periplasm fluid was recovered by an osmotic shock. The pellet from 2 L of WK6 cell culture was resuspended in 30 mL of TES buffer (100 mM Tris\u0026ndash;HCl pH 8.0, 1 mM EDTA, and 20% sucrose) pre-chilled in an ice-water bath. The resuspended cells were incubated for 60 min. After that, 90 mL of pre-chilled deionized water was added, and cells were incubated overnight. Both incubations were carried out on an ice-water bath in a rocking shaker (3D motion). The suspension was centrifuged, and the supernatant was loaded onto a Ni\u003csup\u003e2+\u003c/sup\u003e-NTA-agarose column equilibrated with 20 mM Tris-HCl, 300 mM NaCl, pH 7.5. The elution of the NBs was performed with 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, and pH 7.5. The collected fractions were analyzed by SDS-PAGE and pooled. After that, an extensive dialysis step was performed (4 \u0026deg;C, in a 20 mM Tris-HCl, 300 mM NaCl, pH 7.5). Protein purity was \u0026gt;\u0026thinsp;95%, as evaluated in SDS-PAGE. No aggregation was observed, as inferred from size exclusion chromatography (SEC) analysis. Pure NB was stored at \u0026minus;\u0026thinsp;70 \u0026deg;C until use. When osmotic shock was performed, protein preparations of higher purity were obtained compared with lysis by sonication.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHydrodynamic Behavior of the NB:FXN Protein Complexes\u003c/h2\u003e \u003cp\u003eSEC-FPLC was performed using a Superose-6 column (GE Healthcare). Protein concentration was 6\u0026ndash;10 \u0026micro;M, a volume of 100\u0026micro;L was typically injected, and the running buffer was 20 mM Tris-HCl, 100 mM NaCl, at pH 7.4. The experiment was conducted at room temperature (\u0026sim;25\u0026deg;C) at a 0.5 mL/min flow rate. A JASCO HPLC instrument was used. It had an automatic injector, a quaternary pump, and a UV-VIS UV-2075 (elution was monitored at 280 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThermal Shift Assay\u003c/h2\u003e \u003cp\u003eTemperature-induced denaturation of NB:FXN complexes using the pathogenic G130V FXN variant was monitored by the change in the Sypro Orange dye fluorescence using protein at a 5.0 \u0026micro;M concentration in 50 mM sodium phosphate buffer, pH 7.0. Samples of FXN G130V alone, wild-type FXN alone or NB alone, and samples without any protein were also included as controls. The dye was used at 2 \u0026times; (as suggested by Thermo Fisher Scientific). The temperature slope was 1 \u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (from 20 to 90\u0026deg;C). Excitation and emission ranges were 470\u0026ndash;500 and 540\u0026ndash;700 nm, respectively. The fluorescence signal was quenched in the aqueous environment but became unquenched when the probe was bound to the apolar residues upon unfolding. Experiments by triplicate were carried out in a Step One Real-Time-PCR instrument (Applied Biosystems, CA, U.S.A.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNB Titration by NMR and the NB Interaction Sites on the FXN Surface\u003c/h2\u003e \u003cp\u003e \u003csup\u003e15\u003c/sup\u003eN labeled FXN was prepared as before [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Samples for NMR experiments contained 0.1 mM \u003csup\u003e15\u003c/sup\u003eN-labelled protein in a buffer supplemented with 5% D\u003csub\u003e2\u003c/sub\u003eO. NMR experiments were performed at 22\u0026deg; C in a Bruker 600 MHz Avance III spectrometer equipped with a TXI probe. The NMR data were processed with NMRPipe [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and analyzed using NMRViewJ [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC experiments were performed to follow the interaction by chemical shift perturbations. The \u003csup\u003e15\u003c/sup\u003eN labeled FXN was titrated with each NB at molar ratios of 0:1, 0:33, 0.66, and 1:1 (NB:\u003csup\u003e15\u003c/sup\u003eN-FXN). The experiment was performed in a 25 mM Tris-HC, 150 mM NaCl, pH 7.4 buffer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBiolayer Interferometry Experiments\u003c/h2\u003e \u003cp\u003eThe experiments were carried out using a BLItz instrument (Sartorius). For this experiment, a recombinant FXN H177C variant (\u0026gt;\u0026thinsp;98% pure) was labeled with a bifunctional biotin-maleimide probe of long arm Cat# SP-1501-12 (Vector). Variant FXN H177C-biotin was purified by G25 (separated from the free probe), and extensive dialysis was performed and stored at -70 \u0026deg;C. The sensors were hydrated in TBS buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.4) for these experiments and supplemented with 0.5 mg/mL BSA (TBS-BSA) for 10 minutes. Subsequently, FXN-biotin binding to the streptavidin sensor surface was carried out. Each experiment consisted of a baseline (100 sec, 250 \u0026micro;L), an association (300 sec, 4 \u0026micro;L), and a dissociation (300 sec, 250 \u0026micro;L). After each experiment, the sensor was regenerated by glycine 10 mM pH 1.0 (5 sec, 300 \u0026micro;L) and TBS-BSA buffer washing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture, Treatments, and Materials\u003c/h2\u003e \u003cp\u003eHEK-293T cells (kindly provided by Dr. Ibanez, INQUIMAE, UBA) and Hela Kyoto cells (kindly provided by Dr. Matias Blaustein, iB\u003csup\u003e3\u003c/sup\u003e, UBA) were grown in high glucose (4.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose) Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Natocor), penicillin/streptomycin (100 units mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively, Thermo Fisher Scientific) and 110 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of sodium pyruvate (Thermo Fisher Scientific) in a 37\u0026deg;C humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Polyethylenimine (PEI, PolyAR, UBA) was used for transfection. Briefly, cells were plated (2 \u0026times;10\u003csup\u003e6\u003c/sup\u003e HEK-293T cells per 100 mm plate, 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e HeLa Kyoto cells per well in 12 well plate) and grown for 24 h before transfection. pCMV_MTS_NB_4A7, pCMV_MTS_NB_6B1, pCMV_MTS_NB_16C10, and pCMV_MTS_NB_28F6 vectors encoding each NB (optimized for mammalian cells expression) preceded by the mitochondrial transit signal (MTS) from the citrate synthase enzyme for the mitochondrial matrix localization, were transfected according to the PEI manufacturer\u0026rsquo;s instructions. For the immunofluorescence assays, p-ds-Red2-mito_vector (kindly provided by Dr. Roxana Gorojod, IQUIBICEN, UBA) was co-transfected with the MTS_NB in the same conditions. After transfection, cells were grown for 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE and Western Blotting Assay\u003c/h2\u003e \u003cp\u003eProtein lysates were prepared using RIPA low salt buffer (20 mM Tris-HCl, 150 mM NaCl, 0,1% between 20, 2 mM EDTA, pH 7.40) and complete protease inhibitor solution (Thermo Fisher). Protein concentration was determined using a Bradford reagent (Thermo Fisher). Protein samples (either purified or complete lysates) were boiled in sample buffer (4% SDS, 20% glycerol, 120 mM Tris, pH 6.8, 0.002% bromophenol blue, 200 mM 2-mercaptoethanol) and subjected to 16% SDS-PAGE. Electrophoresis was carried out at room temperature for 20 min at 90V and 1.5h at 150V. Proteins were stained with Coomassie Brilliant Blue G-250. When Western blotting analyses were performed, proteins were transferred to either a PVDF or a nitrocellulose membrane (0.2 \u0026micro;m, Thermo Fisher and BioRad) for 1 h at 100 V. Membranes were blocked for 1 h at room temperature with 5% skimmed milk in 0.05% tween TBS buffer. Blocked membranes were incubated overnight at 4\u0026deg;C with either an anti-human FXN mAb (abcam, ab 110328), an anti-His6 mAb (MA-125, Thermo Fisher), or an anti-GAPDH mAb (SC-47724). Afterward, HRP-conjugated anti-mouse was incubated for 1h at room temperature and visualized by enhanced chemiluminescence (Clarity Substrate, Biorad) using Amersham Imager 680.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAconitase and Succinate Dehydrogenase Activity after NB Transfection\u003c/h2\u003e \u003cp\u003eBriefly, HEK-293T cells were plated (2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per 100 mm plate) and grown for 24 h before transfection (using PEI) with pCMV_MTS_NB_4A7, pCMV_MTS_NB_6B1, pCMV_MTS_NB_16C10, and pCMV_MTS_NB_28F6, according to the PEI manufacturer\u0026rsquo;s instructions (an empty vector control was included). After 48 h, protein lysates were prepared using either PBS buffer or aconitase reaction buffer and complete protease inhibitor (Thermo Fisher). Protein concentration was determined using a Bradford reagent (Thermo Fisher). Succinate dehydrogenase activity was measured using the Succinate Dehydrogenase Activity Assay Kit (Colorimetric) (Abcam; ab228560). Aconitase activity was measured using the Aconitase Enzyme Kit (Abcam, ab109712). All assays were performed following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Oxygen Consumption Rate Measurements after NB Transfection\u003c/h2\u003e \u003cp\u003eThe mitochondrial OCR and ECAR were monitored \u003cem\u003ein vivo\u003c/em\u003e in real-time using a Seahorse XFp analyzer (XFp, Agilent). For this, HEK-293T cells were plated (2 \u0026times;10\u003csup\u003e5\u003c/sup\u003e cells in a 6-well plate) and grown for 24 h before transfection (using PEI) with pCMV_MTS_NB_4A7, pCMV_MTS_NB_6B1, pCMV_MTS_NB_16C10, and pCMV_MTS_NB_28F6, according to the PEI manufacturer\u0026rsquo;s instructions (an empty vector was included as control condition). After 24 h of transfection, 2.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well were plated on pre-coated (with PEI, Sigma) Seahorse XFp Cell Culture Miniplates (Agilent) and maintained in culture conditions for 24 h, when bioenergetics was assessed. Seahorse assays were performed on confluent plates (criterion: cells covering 99.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05% of the plate surface, quantified over a grid of 25 \u0026times; 25 \u0026micro;m). This criterion was confirmed by quantification of H\u0026ouml;escht-positive nuclei at the end of the Seahorse assays (data not shown). On the day of the assay, the culture medium was aspirated and replaced by XF Base Medium (Agilent) supplemented with 25 mM D-glucose, 1 mM sodium pyruvate, and 2mM l-glutamine, pH 7.4. Cells were incubated with this medium for 1 h at 37\u0026deg;C in a non-CO\u003csub\u003e2\u003c/sub\u003e incubator, and then the microplate was loaded into the Seahorse XFp Analyzer (Agilent) following the manufacturer\u0026rsquo;s instructions. Cells were titrated with 0.125 \u0026minus;\u0026thinsp;2.0 \u0026micro;M carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to render the maximum oxygen consumption rate (OCR), and these concentrations were used for experiments. The OCR was determined at the beginning of the assay (basal OCR) and after the sequential addition of 1.0 \u0026micro;M oligomycin (Oligo), 1.0 \u0026micro;M FCCP, and 0.5 \u0026micro;M rotenone plus antimycin A (Rot/AA). Three basal rates and three response rates (after adding a compound) were measured, and the average of these rates was used for data analysis. Respiratory parameters were obtained as follows: Basal respiration is the baseline OCR. Respiration driving proton leak is the OCR after the addition of 1\u0026micro;M Oligo. Respiration driving ATP synthesis is the Basal respiration minus Respiration driving proton leak. Maximum respiration is the OCR after the addition of 1 \u0026micro;M FCCP. Spare respiratory capacity (SRC) is the Maximum respiration minus the Basal respiration.\u003c/p\u003e \u003cp\u003eValues were expressed as a percentage of the OCR corresponding to the last baseline rate (100%). Data was normalized to the cell number at the end of the assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCo-Immunoprecipitation Assay\u003c/h2\u003e \u003cp\u003eHEK-293T cell lysates (500 \u0026micro;g) were incubated overnight at 4\u0026deg;C under agitation with 4 \u0026micro;g of a 6\u0026times;-His tag antibody (MA 1-135, Thermo Fisher). Binding to protein G magnetic beads (Biorad) was performed for 1 h at 4 \u0026ordm;C (previously, beads were washed with the same RIPA buffer). Three washes were performed after binding and then eluted with 25 \u0026micro;L of a 1\u0026times; sample buffer (for SDS-PAGE) containing 8% 2-mercaptoethanol. After that, the co-immunoprecipitated proteins were analyzed by western blotting.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eLC-MS analysis\u003c/h2\u003e \u003cp\u003eFor mass spectrometry analysis, the SDS-PAGE fragments were treated according to LaBaer and coworkers [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. A volume of 4 \u0026micro;L of each sample was injected. Peptide separations were performed on a nano HPLC Ultimate3000 (Thermo Scientific) using a nano column EASY-Spray ES901 (15 cm \u0026times; 50 \u0026micro;m ID, PepMap RSLC C18). The mobile phase flow rate was 300 nL/min using 0.1% formic acid in water (solvent A) and 0.1% formic acid and 100% acetonitrile (solvent B). The gradient profile was set as follows: 4\u0026ndash;30% solvent B for 64 min, 30\u0026ndash;80% solvent B for 7 min and 80% solvent B for 1 min. MS analysis was performed using a Q-Exactive HF mass spectrometer (Thermo Scientific). 1,9 kV of liquid junction voltage and 250\u0026deg;C of capillary temperature were used for ionization. The entire scan method employed a m/z 375\u0026ndash;2000 mass selection, an Orbitrap resolution of 120,000 (at m/z 200), a target automatic gain control (AGC) value of 1e6, and a maximum injection time of 100 msec. After the survey scan, the 15 most intense precursor ions were selected for MS/MS fragmentation. Fragmentation was performed with a normalized collision energy of 28 eV, and MS/MS scans were acquired with a dynamic first mass, AGC target was 5e5, resolution of 30000 (at m/z 200), isolation window of 1.4 m/z units, and maximum IT was 55 ms. Charge state screening was enabled to reject unassigned, singly charged, and equal or more than six protonated ions. A dynamic exclusion time of 25 sec was used to discriminate against previously selected ions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMass Spectrometry Data Analysis\u003c/h2\u003e \u003cp\u003eUsing standardized workflows, MS data were analyzed with Proteome Discoverer (versi\u0026oacute;n 2.4.1.15). Mass spectra *.raw files were searched against a database from Homo sapiens (UP000005640) and NB-16c10 sequence. Precursor and fragment mass tolerance were set to 10 ppm and 0.02 Da, respectively, allowing two missed cleavages. The following modifications were set: -Max. Equal modifications per peptide: 3. Max Dynamic Modifications per peptide: 4. The dynamic modifications included in the analysis were (i) oxidation (+\u0026thinsp;15.995 Da), (ii) N-terminal modification, acetylation (+\u0026thinsp;42.011 Da), (iii) N-terminal modification, Met-loss (-131.040 Da), (iv) N-terminal modification Met-loss plus acetylation (-89.030 Da); the static modification included was the carbamidomethylation (+\u0026thinsp;57.021 Da).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescence Assays\u003c/h2\u003e \u003cp\u003eFor fluorescent reporter assays, cells were plated either into 12-well plates with precision cover glasses or 8-well (Nunc\u0026reg; Lab-Tek\u0026reg; II) coverglass imaging plates. After 48h transfection, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, washed 3 times with PBS, and permeabilized for 5 min with 0.2% Triton X-100 in the same buffer. Blocking was performed for 1 h in 1% BSA solution. After incubation for 1 h with the primary (anti-VHH, rabbit polyclonal antibody) and the secondary antibody (Alexa 488 labeled anti-rabbit antibody, Thermo Fisher) in blocking solution for 1h, cells were extensively washed with PBS, and nuclei were stained with Hoescht dye. Images were captured on an Olympus FV-1000 fluorescence microscope with a 60 \u0026times; oil immersion objective and a Coolsnap HQ2 CCD camera (Photometrics).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eAssembly of the NB:FXN Complexes\u003c/h2\u003e \u003cp\u003eFor structural purposes, FXN:NB_16C10, FXN:NB_4A7 and FXN:NB_6B1 complexes were assembled by incubation and then copurified by using the His-tag present in the nanobodies. Briefly, the complexes were copurified by HisTrap FF column using an imidazole gradient from 20 mM to 500 mM in 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 1 mM DTT. The complexes were eluted at 130 mM of imidazole. The eluted fractions containing the desired complexes were identified using SDS-PAGE, and concentrations were determined by NanoDrop. All the proteins were collected and dialyzed for imidazole removal using a Spectra/PorTM 1RC dialysis membrane (of 6\u0026ndash;8 kDa MWCO). The resulting protein solutions were concentrated on an Amicon Ultra-4 Centrifugal Filter (MWCO 10kDa; Millipore Sigma, Burlington, MA). The final concentration for FXN:NB_16C10, FXN:NB_4A7, and FXN:NB_6B1 was 7.5mg/mL, 3.5mg/mL, and 11mg/mL, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eNB:FXN Crystallization Experiments\u003c/h2\u003e \u003cp\u003eInitial crystallization screening using the copurified complexes was performed at 298K using the sitting-drop vapor-diffusion method with an available collection of commercial conditions. The drops consisted of 0.2\u0026micro;L of protein solution (7.5 mg/mL, 3.5 mg/mL or 11mg/mL of protein complex in a buffer containing 30 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM DTT) and 0.2 \u0026micro;L well solution, and were equilibrated against 60uL of well solution. The extensive screening rendered crystals in several conditions and their corresponding diffraction quality was checked on the beamline BL13-XALOC at the ALBA Synchrotron (Barcelona, Spain). Best diffracting crystals were subsequently scaled up and optimized, yielding crystals grown in 15% PEG 10K, 0.1M ammonium acetate and 0.1 M Bis-Tris pH 5.5 (FXN:NB_16C10); 0.2M Na thiocyanate pH 6.9, 20% PEG 3350 (FXN:_NB4A7); and 0.1 M HEPES pH 7.5, 0.8 M potassium sodium tartrate (FXN:NB_6B1).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eX-ray Data Collection and Processing\u003c/h2\u003e \u003cp\u003eAll data were collected from frozen crystals at 100 K with the PILATUS 6M detector at beamline XALOC (ALBA Synchrotron, Barcelona, Spain). Data processing and scaling were accomplished using XDS [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], POINTLESS, and AIMLESS [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] as implemented in autoPROC [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Statistics for the crystallographic data and structure solution are summarized in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eCrystal Structure Solution, Model Building and Refinement\u003c/h2\u003e \u003cp\u003eThe NB:FXN structures were solved by the molecular-replacement method, as implemented in the program PHASER [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] using \u003cem\u003eab initio\u003c/em\u003e models given by AlphaFold [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] as search models. Then, its initial models were subjected to iterative cycles of model building and refinement with Coot [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] and REFMAC [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], respectively. Final refinement cycles were performed with PHENIX [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], yielding the refinement and data collection statistics summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Figures were generated using PyMOL [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and ChimeraX [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The final refinement parameters are summarized in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSmall Angle X-ray Scattering (SAXS)\u003c/h3\u003e\n\u003cp\u003eSAXS experiments were performed at the beamline B21 of the Diamond Light Source [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Samples of 45 \u0026micro;L of all the complexes at different concentrations were loaded onto an SRT-C SEC-300 (Sepax) column equilibrated in buffer (20 mM Tris-HCl pH 8.0 and 150 mM NaCl) and connected to an Agilent 1200 HPLC system at 18\u0026deg;C. The continuously eluting samples were exposed for 3s in 10s acquisition blocks using an X-ray wavelength of 1 \u0026Aring; and a sample-to-detector (Eiger 4M) distance of 3.7 m. The data covered a momentum transfer range of 0.0032\u0026thinsp;\u0026lt;\u0026thinsp;q\u0026thinsp;\u0026lt;\u0026thinsp;0.34 \u0026Aring;\u0026minus;1. The frames recorded immediately before the sample elution were subtracted from the protein scattering profiles. The Sc\u0026aring;tter software package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.bioisis.net\" target=\"_blank\"\u003ewww.bioisis.net\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.bioisis.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze data, buffer-subtraction, scaling, merging, and checking possible radiation damage of the samples. The Rg value was calculated with the Guinier approximation, assuming that at very small angles q\u0026thinsp;\u0026lt;\u0026thinsp;1.3/Rg. The particle distance distribution, Dmax, was calculated from the scattering pattern with GNOM, and shape estimation was carried out with DAMMIF/DAMMIN; all these programs are included in the ATSAS package [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The protein molecular mass was estimated with GNOM. Interactively generated PDB-based homology models were made using the program COOT by manually adjusting the X-ray structures obtained in this work, into the envelope given by SAXS until a good correlation between the real-space scattering profile calculated for the homology model matched the experimental scattering data. This was computed with the program FoXS [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eSequence Analysis and Structure Predictions of the NB/FXN Complexes\u003c/h2\u003e \u003cp\u003eAfter the phage display selection using native mature FXN as the target, and a preliminary L-Cys-desulfurase activity screening, a total of 30 NBs specific for human FXN were initially sequenced. We found a substantial sequence diversity for the complementary determining regions (CDRs) CDR1, CDR2, and CDR3 (Figs.\u0026nbsp;1\u003cb\u003eD and 2\u003c/b\u003e), yielding 16 different sequences. Eleven NBs were chosen for protein expression in \u003cem\u003eE. coli\u003c/em\u003e WK6 strain, purification, and \u003cem\u003ein vitro\u003c/em\u003e studies. Four NBs (NB_4A7, NB_6B1, NB_16C10, and NB_28F6, \u003cb\u003eTable S2\u003c/b\u003e) and the corresponding NB:FXN complexes were deeply studied in this work. Alpha-Fold 3 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] were used to infer structures of NB:FXN complexes from their amino acid sequences. The results suggested many different types of complexes NB:FXN might be formed (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). More than one different NB:FXN complex type was usually predicted for each NB sequence. Furthermore, predictions indicated that some NBs might display an inhibitory effect because the binding region comprises a surface of FXN involved in the supercomplex architecture (e.g., an ISCU2 or NFS1 interacting surface). On the other hand, some NBs might interact with FXN surface areas that are not involved in inter-subunit interactions. In those cases, NBs may not affect the activation exerted by FXN. However, NBs may fix a particular conformation, altering the internal motions of the FXN and, therefore, modulating the FXN function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eNB Expression in E. coli WK6\u003c/h2\u003e \u003cp\u003eNBs were purified by NTA-Ni\u003csup\u003e2+\u003c/sup\u003e, yielding 95% pure protein (5\u0026ndash;15 mg/L). In all cases, monomeric conformations were obtained, as judged by SEC-FPLC (see below). For NB_28F6, protein degradation was observed when the NB was stored at 4\u0026deg;C. Thus, we decided to preserve the NBs at -70\u0026deg;C. Apart from NB_28F6, which exhibited proteolysis, the masses of the purified NBs were in the 2 Da range of the expected masses, as deduced from the amino acid sequences, considering the predicted processing site after NB export to the periplasmic space (the processing of the signal peptide results in an N-term QVQLQ, \u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e and \u003cb\u003eTable S2\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eNB Effect on Supercomplex Activity in vitro\u003c/h2\u003e \u003cp\u003eAfter nanobody purification, we investigated the effect of NBs addition to the enzymatic reaction on the L-Cys desulfurase catalysis \u003cb\u003e(Figs.\u0026nbsp;3A and S2)\u003c/b\u003e. The NBs NB_4A7 and NB_16C10 exhibited a low-middle degree of inhibition, while NB_6B1 and NB_28F6 showed higher inhibition of the \u003cem\u003ein vitro\u003c/em\u003e L-Cys-desulfurase activity. It is worthy of note that the complex (NFS1/ACP-ISD11/ISCU)\u003csub\u003e2\u003c/sub\u003e shows lower activity than the core, (NFS1/ACP-ISD11)\u003csub\u003e2\u003c/sub\u003e. In fact, one can infer ISCU interaction with (NFS1/ACP-ISD11)\u003csub\u003e2\u003c/sub\u003e from the observed decrease in the desulfurase activity [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Higher NB concentrations of NB_6B1 and NB_28F6 produced the highest decrease in the activity of the supercomplex like the one detected in the virtual absence of FXN, which represents a drop in the value corresponding to (NFS1/ACP-ISD11/ISCU)\u003csub\u003e2\u003c/sub\u003e. On the other hand, the inhibitory effect of higher concentrations of NB_16C10 was lower than the observed for NB_6B1 and NB_28F6, suggesting that NB_16C10 affects the conformation or the topology of the supercomplex but would not interfere with the FXN-supercomplex interaction in these experimental conditions. The activity of the supercomplex in the presence of the higher level of NB_4A7 was the highest observed among the NBs studied \u003cem\u003ein vitro\u003c/em\u003e in this condition \u003cb\u003e(Fig.\u0026nbsp;3B-E)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eIn Vitro Characterization of the NB:FXN Interaction\u003c/h3\u003e\n\u003cp\u003eTo evaluate the \u003cem\u003ein vitro\u003c/em\u003e NB:FXN complex formation, we first studied the interaction by size-exclusion chromatography (SEC). Purified NBs, FXN, or the mix of both proteins (injection after a 10-minute incubation of FXN and NB at room temperature) were loaded in an analytical SEC system. As judged by the SEC profiles, many of the selected NBs against FXN formed stable complexes (Figs.\u0026nbsp;4 \u003cb\u003eand S3\u003c/b\u003e), and their profiles suggested a slow binding equilibrium. Besides this, the SEC profile corresponding to NB_15C5 was compatible with the absence of complex formation exhibiting two peaks consistent with free FXN and free NB (\u003cb\u003eFigure S3C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe binding between NBs and FXN was studied employing biolayer interferometry (BLI). To evaluate the interaction, a biotin-labeled FXN variant (H177C, \u003cb\u003eFigure S4\u003c/b\u003e) was immobilized in a streptavidin sensor at 10 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The selected NBs exhibited high affinity with equilibrium dissociation constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e in the nanomolar range 1\u0026ndash;33 nM, and association and dissociation kinetic rate coefficients \u003cem\u003ek\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003ed\u003c/sub\u003e in the range 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M, respectively (Fig.\u0026nbsp;4 and \u003cb\u003eTable S3\u003c/b\u003e).\u003c/p\u003e\n\u003ch3\u003eIdentification of the NB Binding Site on the FXN Surface\u003c/h3\u003e\n\u003cp\u003eThe binding sites of this subset of NBs were studied with a resolution at the level of the amino acid residues by NMR, analyzing the chemical shift perturbations (CSP). FXN was produced in \u003cem\u003eE. coli\u003c/em\u003e using the \u003csup\u003e15\u003c/sup\u003eN source \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl and purified for these experiments. The target was then titrated with each of the NBs, and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN-HSQC NMR bidimensional spectra were acquired. Then, CSP and changes in the cross-peak intensities were analyzed (Figs.\u0026nbsp;5 and \u003cb\u003eS5-S8\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eCSP results indicated that NBs NB_4A7, NB_6B1, and NB_16C10 bind to a similar site on the FXN surface (residues involved are shown in \u003cb\u003eTable S4)\u003c/b\u003e. Instead, NB_28F6 presents a different binding mode, as indicated by the residues involved in the interaction. As judged by the discontinuous pattern of CSP observed for almost all the cross peaks involved, the results suggested, in all cases, a binding mechanism characterized by a slow dissociation equilibrium. Remarkably, some residues from the FXN core exhibited significant CSP, including aromatic side chains Phe110, Tyr123, and Phe127 and the aliphatic residue Leu113. This suggests that the interaction between NB and FXN may adjust some conformational details of the FXN structure. In the case of NB_28F6, some residues far from the suggested interaction site also exhibited high CSP (\u003cb\u003eTable S4\u003c/b\u003e). Among them, Tyr143, Ile145, and Trp155, the latter involved in the assembly site of the supercomplex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eModulation of the FXN Conformational Stability by NB Interaction\u003c/h2\u003e \u003cp\u003eTo test whether NB interactions could stabilize the FXN conformation, we conducted \u003cem\u003ein vitro\u003c/em\u003e temperature-induced unfolding experiments. Protein denaturation was monitored by the fluorescence of the Sypro-orange probe. Briefly, when the dye interacts with the unfolded state of proteins, the quantum yield increases, exhibiting an increase in the fluorescence intensity. We used FXN G130V as a probe for highly unstable Friedreich\u0026rsquo;s Ataxia (FRDA) variants [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] for these experiments. This variant is unstable \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, giving rise to very low FXN concentrations in the mitochondrial matrix \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNoteworthy, the NBs were able to significantly stabilize the G130V FXN variant as judged by a significant shift (\u0026ge; 17 \u0026deg;C) in the observed Tm values (e.g., Tm values are 51.8 \u0026plusmn; 0.3 and 70.1 \u0026plusmn; 0.1\u0026deg;C, for the G130V variant and NB_4A7: G130V_FXN complex, respectively, \u003cb\u003eFigs.\u0026nbsp;6A and E\u003c/b\u003e). The Tm value corresponding to the complex is even higher than that observed for the wild-type FXN (Tm\u0026thinsp;=\u0026thinsp;65.9\u0026plusmn;0.3\u0026deg;C). Similar results were obtained for the rest of the NBs (Fig.\u0026nbsp;6\u003cb\u003e)\u003c/b\u003e, suggesting that they might stabilize the native conformation of FXN in the cellular environment. On the other hand, Sypro-orange dye did not show significant interaction with the nanobodies used in this assay in this range of temperature (blue lines in \u003cb\u003eFig.\u0026nbsp;6 A-D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eThree-dimensional Structure of NB:FXN Complexes\u003c/h2\u003e \u003cp\u003eTo get high-resolution information on the binding mode of the NBs studied in this work, NB_4A7, NB_6B1, and NB16C10 in complex with wild-type FXN (90\u0026ndash;210 variant) were crystallized, and their structures solved (Fig.\u0026nbsp;7). The three crystallographic complexes were solved at atomic resolutions of 1.25 for FXN: NB_4A7, 1.48 for FXN: NB_6B1 complex, and 2.0 \u0026Aring; for FXN: NB_16C10 complex. The quality of the electron density map was excellent (\u003cb\u003eFigure S9\u003c/b\u003e), allowing clear modeling of both proteins and fully identifying the residues involved in protein-protein interaction. FXN residues involved in the interaction are listed in \u003cb\u003eTable S5\u003c/b\u003e. The structure of human FXN in our complexes presents a nearly identical structure to that reported previously (PDB ID: 1EKG) [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], with RMSD values ranging from 0.21\u0026ndash;0.24 \u0026Aring; (for superimposition of all Cα atoms). FXN presents two parallel α-helices supported by a platform provided by a five-stranded, antiparallel β-sheet. The NBs adopt the typical immunoglobulin fold, with ten β-strands forming two β-sheets connected by loops and a conserved disulfide bond between Cys22 and Cys95 (Fig.\u0026nbsp;2\u003cb\u003eA\u003c/b\u003e). The NB:FXN crystal structures confirmed the interaction surface and binding regions observed by NMR experiments (CSP results, \u003cb\u003eFig.\u0026nbsp;5\u003c/b\u003e and \u003cb\u003eTable S4\u003c/b\u003e). In all cases, the two proteins in the complex are arranged in a T fashion mode with FXN orienting the acidic ridge (the loop linking α1 and β1, residues Glu114-Glu122), the loop β2\u0026ndash;β3 (residues 135\u0026ndash;139) and the tip of helix α1, against the central part of the NB β-sandwich. Specifically, the NBs interact with frataxin through the loop β3\u0026ndash;β4 (residues Arg38-His46, using \u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e numbering), the β4-strand (residues Leu47-Arg50), and the loop β5\u0026ndash;β6 (residues Asp61-Lys64); NB_4A7 and NB_16C10 also interact through Asn58 from β4 (Fig.\u0026nbsp;7). Remarkably, the CDR regions of the NBs are not directly involved in binding to FXN. Some relevant electrostatic interactions are observed in the NB:FXN complex (e.g., Glu121\u003csub\u003eFXN\u003c/sub\u003e-Arg38\u003csub\u003eNB\u003c/sub\u003e, Lys135\u003csub\u003eFXN\u003c/sub\u003e-Asp61\u003csub\u003eNB\u003c/sub\u003e, Asp139\u003csub\u003eFXN\u003c/sub\u003e-Lys64\u003csub\u003eNB\u003c/sub\u003e, Phe120\u003csub\u003eFXN\u003c/sub\u003e-Arg50\u003csub\u003eNB\u003c/sub\u003e) (Fig.\u0026nbsp;7), involving a molecular contact surface area of 581.7 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e. Structural comparison of FXN alone and in complex with NB (\u003cb\u003eFigure S10\u003c/b\u003e) reveals that major changes upon NB interaction are in Loop 1, L1, connecting α1\u0026ndash;β1, and Loop 2, L2, connecting β2\u0026ndash;β3 (Fig.\u0026nbsp;7 and \u003cb\u003eFigure S10\u003c/b\u003e). While some variations are also observed in β5\u0026ndash;β6 turn and the loop connecting β6 and α2, depending on the NB. Overall, a similar pattern of interactions is conserved for the interaction of FXN and NB_4A7, NB_6B1, and NB_16C10, where loops L1 and L2 and a punctual interaction through α2 are pivotal for protein-protein recognition.\u003c/p\u003e \u003cp\u003eD122Y and G137V mutations in FXN (two pathogenic FRDA variants found in patients) occur in residues located on the interface of the NB:FXN complexes, (\u003cb\u003eFigure S11\u003c/b\u003e). Whether these NBs can interact and stabilize a highly unstable variant as G137V [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] should be investigated. Whereas this pathogenic FXN variant activated the supercomplex and did not exhibit structural alterations, it shows a significant decrease in its conformational stability that is correlated to the very low FXN concentration found in people living with FRDA, considered the main feature of the physiopathology of this variant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eSAXS studies on FXN:16C10 and FXN:28F6 complexes in solution\u003c/h2\u003e \u003cp\u003eThe complex FXN:NB_28F6 did not produce crystals in our hands; thus, we decided to perform Small Angle X-ray Scattering (SAXS, \u003cb\u003eTable S6\u003c/b\u003e) studies to check if the interaction would follow the same mode observed for NBs NB_16C10, NB_6B1 and NB_4A7. The complex FXN:NB_16C10 was also measured for comparison with our crystal structures. In both cases, we observed a good superimposition between the experimental and theoretical scattering curves (Fig.\u0026nbsp;8\u003cb\u003eA\u003c/b\u003e). As expected, the FXN:NB_16C10 complex, the low-resolution envelope in solution, fits with the crystal structure of the complex in which the two proteins are oriented in a T-fashion mode (Fig.\u0026nbsp;8\u003cb\u003eD\u003c/b\u003e). However, the FXN:28F6 complex shows a new disposition in which the two proteins pack in a parallel fashion with the b-sheets from both proteins configured as a single long b-sheet (Fig.\u0026nbsp;8\u003cb\u003eC\u003c/b\u003e). The model observed in the solution for the FXN:NB_28F6 complex is compatible with the predicted arrangement provided by AlfaFold3 (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD\u003c/b\u003e). As discussed below, this different arrangement for the NB_28F6 can explain the observed differences in the behavior in our cellular experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003eHow the NB:FXN Complex Docks onto the Supercomplex Structure\u003c/h2\u003e \u003cp\u003eWhether the selected NBs might block the interaction of FXN with the rest of the supercomplex depends on the specific structure of the NB:FXN complex and how it fits/perturbs into the supercomplex structure throughout the catalytic cycle. To gain structural information on that hypothesis, we combined information provided by X-ray diffraction experiments concerning NB:FXN complexes (from this work) with previous structural data regarding the supercomplex (NFS1/ACP-ISD11/ISCU/FXN)\u003csub\u003e2\u003c/sub\u003e provided by cryo-electron microscopy (PDB ID: 6NZU [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]). The NB:FXN structures \u003cb\u003e(Fig.\u0026nbsp;9A)\u003c/b\u003e were docked onto the supercomplex by the superimposition of the FXN subunit. In the type I NB:FXN binding (NBs NB_4A7, NB_6B1, and NB_16C10, \u003cb\u003eFig.\u0026nbsp;9B\u003c/b\u003e), only slight clashes between the NB and the rest of the subunits of the supercomplex were predicted. The contacts that the NB may establish through its β3\u0026ndash;β4 connector with one of the NFS1 subunits might lead to the inhibitory effects observed \u003cem\u003ein vitro\u003c/em\u003e. In the case of NB_16C10, residues involved in the clashes may be Pro41\u003csub\u003eNB\u003c/sub\u003e-Asn245\u003csub\u003eNFS1\u003c/sub\u003e, Gly42\u003csub\u003eNB\u003c/sub\u003e-Arg271\u003csub\u003eNFS1\u003c/sub\u003e, Lys43\u003csub\u003eNB\u003c/sub\u003e-Glu120\u003csub\u003eNFS1\u003c/sub\u003e, Arg45\u003csub\u003eNB\u003c/sub\u003e-Arg273\u003csub\u003eNFS1\u003c/sub\u003e, and His46\u003csub\u003eNB\u003c/sub\u003e-Arg273\u003csub\u003eNFS1\u003c/sub\u003e (using the numbering shown in \u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e for the NB and the numbering used in PDB ID: 6NZU for NFS1). Whether the plasticity of the protein chains might properly accommodate this stretch could depend on the amino acid sequence of the NB β3\u0026ndash;β4 connector (Fig.\u0026nbsp;9\u003cb\u003eB\u003c/b\u003e), thus determining the rigidity/flexibility behavior and, ultimately, the inhibitory power of these NBs that bind using the same site. Given that NB_4A7 and NB_16C10 have an extra Gly in that stretch, we expect more flexibility than in the case of NB_6B1, in which an Asn residue occupies the place of the Gly44. The edition by reengineering β3\u0026ndash;β4 connectors will be a key for obtaining better FXN binders.\u003c/p\u003e \u003cp\u003eRemarkably, when the supercomplex formed by NFS1, ACP-ISD11, ISCU2, and FXN subunits was loaded in SEC-FPLC system, in the presence of NB_4A7, the elution of the complete assembly shifted to a lower elution time (from 37.65 to 37.28 min, black arrow 2 to 3, absence or presence of NB_4A7) (Fig.\u0026nbsp;9\u003cb\u003eC\u003c/b\u003e). By contrast, when FXN was absent, the presence of NB_4A7 did not modify the elution time of the subcomplex (NFS1/ACP-ISD11/ISCU) (Fig.\u0026nbsp;9\u003cb\u003eC\u003c/b\u003e, elution time 38.4 min, black arrow 1 dark and light green, with or without NB_4A7), a fact indicative of the NB specificity for FXN. More importantly, these results suggest that NB_4A7 can bind FXN in the context of the supercomplex. Additionally, it was evident that a fraction of the NB bound to the free FXN shifted its elution time to lower values, indicating the stabilization of the complex NB:FXN (elution time shifted from 44.4 to 43.3 min in the absence or presence of NB_4A7, respectively). It is worthy of note that under these experimental conditions, the interaction of FXN with NFS1 is not so strong; in fact, other research groups reported a dissociation constant for wild-type FXN of \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e ~3\u0026plusmn;1 \u0026micro;M [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, we expected a high fraction of unbound FXN (\u0026sim;55% unbound, \u0026sim;45% bound [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]). Moreover, considering the dilution due to the SEC column volume, the unbound FXN fraction may be higher (a half dilution would result in \u0026sim;72% unbound and \u0026sim;28% bound).\u003c/p\u003e \u003cp\u003eWe also carried out an interferometry experiment to investigate whether supercomplex and NB_4A7 can simultaneously bind to FXN (Fig.\u0026nbsp;9\u003cb\u003eD\u003c/b\u003e). In this experiment, streptavidin sensors were sensitized using the biotin-labeled FXN H177S variant, (\u003cb\u003eFigure S4\u003c/b\u003e), and after that, sensors were incubated with solution containing both NB and the subcomplex (NFS1/ACP-ISD11/ISCU2)\u003csub\u003e2\u003c/sub\u003e, in the presence of a 3:1 excess of ISCU2: NFS1/ACP-ISD11/ISCU2)\u003csub\u003e2\u003c/sub\u003e, L-Cys and PLP to increase the affinity between ISCU2 and the core complex (NFS1/ACP-ISD11)\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Our results showed that the signal corresponding to the complete mix of proteins is even higher than the observed for the NB_4A7 alone, suggesting that the interaction of FXN with the NB_4A7 does not impede the supercomplex formation. Additionally, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e measured for FXN: (NFS1/ACP-ISD11/ISCU2)\u003csub\u003e2\u003c/sub\u003e. interaction was \u0026sim;0.6 \u0026micro;M, a value in the range of the dissociation constant previously measured under similar conditions (0.2 \u0026micro;M) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, when a similar experiment was carried out using NB_28F6 (Fig.\u0026nbsp;9\u003cb\u003eE\u003c/b\u003e), which binds to the helix 1, the signal observed for the subcomplex (NFS1/ACP-ISD11/ISCU2)\u003csub\u003e2\u003c/sub\u003e and NIAI\u0026thinsp;+\u0026thinsp;NB_28F6 was similar to that observed for NB_28F6. Suggesting that this NB inhibits the binding of the subcomplex to FXN, in agreement with structural results (SAXS and NMR).\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of NBs in the Mitochondria of Human Cell lines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the effects of NB:FXN interaction on the cellular metabolism, we first studied whether the NBs could be successfully imported to the mitochondrial matrix when expressed in human cell lines. We used transfected HeLa Kyoto cells to evaluate this by immunofluorescence. We prepared vectors that included the NB sequence in frame with the citrate synthase mitochondrial transit sequence (MTS) for mitochondrial matrix localization. The cells were co-transfected with the \u003cem\u003eDiscosoma\u003c/em\u003e red fluorescent protein (dsRed Mito), which also included an MTS (cytochrome c oxidase subunit VIII). The transfected cells were analyzed after 48 hours post-transfection.\u003c/p\u003e \u003cp\u003eAs expected, when NBs were detected using an anti-VHH polyclonal serum, which recognizes the NBs invariable regions (followed by an anti-rabbit secondary antibody labeled with Alexa 488), the NB signal co-localized with that obtained with dsRed-Mito, strongly suggesting a mitochondrial localization of the NBs. This behavior was observed for the four nanobodies studied in this work (Fig.\u0026nbsp;1\u003cb\u003e0\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eImportantly, the expression of NBs NB_4A7, NB_6B1 and NB_16C10 did not alter the viability of the cells (\u003cb\u003eFigure S12\u003c/b\u003e). Therefore, we demonstrated that the NBs are localized in the mitochondria and that their expression did not significantly affect the cell viability. In turn, even though the expression of NB_28F6 was very low, it was possible to evaluate its subcellular localization, and the analysis suggests that it is in mitochondria. On the other hand, the viability analysis of cells transfected with NB_28F6 should be examined cautiously because of the lower expression levels and the possibility that the effects were not comparable to the rest of the NBs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNB Expression and Interaction between FXN and NB in HEK-293T Cells. Characterization of Fe-S clusters related to mitochondrial bioenergetics\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCo-immunoprecipitation was used to investigate whether the NBs can interact with FXN in a cellular environment. The NB candidate, NB_16C10 in this case, was expressed in HEK-293T cells, and its interaction with endogenous FXN was explored. Noteworthy, endogenous FXN was co-immunoprecipitated with NB_16C10 using an anti-Histag antibody, which indicated that a stable NB:FXN complex was formed in the cells (Fig.\u0026nbsp;1\u003cb\u003e1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, samples corresponding to the co-immunoprecipitation were investigated by mass spectrometry. The analysis of the peptides corresponding to the recovered proteins indicated a strong interaction between the NB_16C10 and FXN (\u003cb\u003eTable S7\u003c/b\u003e). Furthermore, peptides corresponding to citrate synthase suggested that the NB precursor containing the MTS of this mitochondrial enzyme can interact with FXN in the cellular environment. This result points to future investigations concerning whether the NB and FXN could interact in the cytosol or, conversely, the precursor of the NB might interact with FXN in the mitochondrial matrix.\u003c/p\u003e \u003cp\u003eRemarkably, no proteins other than FXN, NB, and those recovered when cell samples were transfected with the empty vector were consistently retrieved, suggesting that the method used for cellular lysis may perturb the stability of other complexes involving protein-protein interactions of lower affinities than that of NB:FXN (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e for FXN in the nanomolar range, \u003cb\u003eTable S3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe effect of NB expression on FXN expression was studied. The densitometric analysis of western blot membranes (three independent experiments) showed similar levels of FXN among the cells transfected with an empty vector (\u003cb\u003eFigure S13A\u003c/b\u003e). Besides this, the expression of NB_4A7, NB_6B1, and NB_16C10 in HEK-293T cells was substantial (\u003cb\u003eFigure S13B\u003c/b\u003e). However, the expression of NB_28F6 was significantly lower (\u003cb\u003eFigure S13B\u003c/b\u003e) than the others (~\u0026thinsp;90% lower, as evidenced by western blotting).\u003c/p\u003e \u003cp\u003eTransfected HEK-293T cells were evaluated to identify possible adverse effects of the NB expression on [Fe-S] cluster-dependent enzymatic activities from aconitase (ACO) and succinate dehydrogenase (SDH). The expression of NBs showed only slight modulations of ACO and SDH activities (Figs.\u0026nbsp;1\u003cb\u003e2 A and B)\u003c/b\u003e. Only the expression of the NB_16C10 led to a slight decrease of the SDH enzyme in HEK-293T cells (Fig.\u0026nbsp;1\u003cb\u003e2 B)\u003c/b\u003e. More experiments will be carried out to evaluate whether this is a direct effect on the enzyme through the alteration of Fe-S cluster assembly activity or, on the other hand, the modulation of SDH is the consequence of more complex processes involving transcription or/and translation, or even post-translational modifications.\u003c/p\u003e \u003cp\u003eTo gain a more global picture of the effect of the FXN_ NB expression in these cells, we measured the oxygen consumption rate (OCR) 48h after transfection (Figs.\u0026nbsp;1\u003cb\u003e2 C-F\u003c/b\u003e), which is indicative of the oxidative phosphorylation (OXPHOS) efficiency. Our results suggested that the OCR is not altered when NB_6B1 or 16C10 are expressed. These NBs exhibited similar extents in the increase of the OCR after FCCP treatment, revealing a similar maximal respiratory capacity (Fig.\u0026nbsp;1\u003cb\u003e2G\u003c/b\u003e), as non-transfected cells. On the other hand, NB_4A7 exhibited a slight decrease in the basal respiratory capacity, possibly compatible with the slight decrease of SDH (Complex II, not significant) activity. However, the observed metabolic changes seem subtle, given the considerable mitochondrial protein expression of NB_4A7, 6B1, and 16C10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn turn, when the HEK-293T cells were transfected with a vector encoding NB_28F6, even though the expression of this NB was significantly lower than that of the other NBs (\u003cb\u003eFigure S13B\u003c/b\u003e), a decrease in basal and maximal respiration was observed (Fig.\u0026nbsp;1\u003cb\u003e2F\u003c/b\u003e), indicative of decreased mitochondrial metabolism.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this work, we characterized new molecular tools to explore if it is possible to modulate FXN stability and function through its interaction with foreign tutor proteins. With this aim, twenty NBs specific to FXN were selected by phage display. We found that NBs can increase FXN conformational stability. Binding was in the nanomolar range of 1\u0026ndash;33 nM, suggesting a strong interaction, a key feature for function modulation purposes. SEC, NMR, and BLI results also suggested a slow dissociation equilibrium. In addition, \u003cem\u003ein vitro\u003c/em\u003e L-Cys-desulfurase activity could be modulated by the NBs; while 1:1 molar ratio does not affect L-Cys desulfurase activity for some NBs (NB_4A7, 5A8, 29F7, and 83G4, \u003cb\u003eFigs.\u0026nbsp;3 and S2\u003c/b\u003e), an increase to 1:5 ratio was inhibitory. As at 1:1 ratio, \u0026sim;83% of the FXN is in complex with NB_4A7 (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e=33.0 nM), whereas \u0026sim;17% is free (calculated using the ligand binding simulation tool [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]), the inhibition observed at 5:1 indicates a more complex interaction of the NB with the rest of the proteins of the system, suggesting unspecific binding under these conditions. The relevance of this fact will be studied further. On the other hand, binding of NB_4A7 to FXN did not inhibit the interaction of FXN with the supercomplex (Fig.\u0026nbsp;9), in agreement with the preservation of the \u0026sim;85% of Cys desulfurase activity observed \u003cem\u003ein vitro\u003c/em\u003e for 1:1 ratio (Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eDifferent experimental techniques (NMR, X-ray crystallography, and SAXS) enabled us to characterize the interaction between the FXN and the NBs. Two distinct types of binding, which we have designated as Type I binding (as we find for NBs NB_16C10, NB_6B1, and NB_4A7), and Type II binding (as found for NB_28F6) were found (Figs.\u0026nbsp;5, \u003cb\u003e7, and 8\u003c/b\u003e). The complexes with a Type I binding have two key polar interactions with the FXN residues of the loop L1 of FXN, specifically, the residues Glu121, Pro117, and Thr119, involving a molecular contact surface area of 581.7 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e. Residue Glu121 plays an essential role in the protein interaction of Type I by making a salt bridge interaction with the residue Arg38 and polar interactions with Ser62 of the NBs. Pro117 and Thr119 also play an important role by establishing H-bonds with residues His46 and Leu47 of β5 from the NBs. Even though, Asn52 from CDR2 makes an H-bond with backbone Gly138 from FXN, and Val98, Pro99, Pro100, from CDR3 are at Van der Waals distance of FXN, in Type I interaction mode, the recognition is not made using the three segments CDR1, CDR2, and CDR3. However, this is common for NBs that use a far vaster diversity of structural stretch combinations to bind antigens [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo study the effect of NBs \u003cem\u003ein vivo\u003c/em\u003e, human cell lines were transfected with vectors encoding four different NBs. Three presented a considerable expression level; instead, NB_28F6 exhibited significantly lower expression. The four transfected NBs were found in the mitochondria of Hela Kyoto cells. Remarkably, the expression of the NBs in HEK-293T and HeLa Kyoto cells did not alter FXN expression or cell viability.\u003c/p\u003e \u003cp\u003eConsidering the degree of modulation of L-Cys desulfurase \u003cem\u003ein vitro\u003c/em\u003e, the higher expression level of NBs in the cells, and the plausible broader spectrum of functions that FXN might exert inside the cells, including direct interactions with iron, aconitase [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], superoxide dismutase [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e] or Complex I from the respiratory chain [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], we reasoned that the expression of the NBs could affect the Fe-S cluster-dependent enzymatic activities and energetics. However, the expression of NB_6B1 and NB_4A7 did not have effects over Fe-S dependent ACO and SDH activities, and the expression of NB_16C10 neither had effects over ACO but exhibited a slight inhibition over SDH (Fig.\u0026nbsp;1\u003cb\u003e2\u003c/b\u003e). This last fact could be compatible with the absence of \u003cem\u003ein-cell\u003c/em\u003e inhibition of mitochondrial L-Cys desulfurase NFS1 supercomplex in HEK-293T cells overexpressing NBs. Moreover, in a more global picture of the mitochondrial metabolism, we demonstrated that the expression of NB_4A7, NB_6B1 and NB_16C10 in HEK-293T did not alter the mitochondrial respiration 48 h after transfection, suggesting that the mitochondrial global metabolism is not significantly perturbed when these NBs are expressed, imported into the mitochondria and eventually interacting with FXN (as judged by co-immunoprecipitation assay).\u003c/p\u003e \u003cp\u003eRemarkably, NB_28F6 yielded a similar profile of ACO and SDH activities. However, under the same conditions, NB_28F6 showed very poor expression levels (\u0026sim;5 times lower in average, \u003cb\u003eFigure S13\u003c/b\u003e). Thus, we could not evaluate its effect on metabolism with confidence. The observed decrease of the OCR (even with a considerably lower protein expression level than the other NBs) suggests that NB_28F6 expression affects mitochondrial OCR through FXN intervention. In particular, the binding surface of FXN involved in the interaction with NB_28F6 could explain the inhibitory effects observed \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Figs.\u0026nbsp;3 \u003cb\u003eand 13\u003c/b\u003e). As evidenced by our NMR results, it involves a larger portion of helix α1 in FXN, the acidic ridge of FXN and NB_28F6 binding resulted in the inhibition of the FXN-supercomplex interaction (Fig.\u0026nbsp;9). Unfortunately, the crystal structure of the FXN: NB_28F6 complex could not be obtained. We consider that some heterogeneity in the sample due to partial proteolysis of this NB (mass spectrometry results) might impede the crystallization process.\u003c/p\u003e \u003cp\u003eThese different results between the \u003cem\u003ein vitro\u003c/em\u003e and in-cell effects can be understood considering that the \u003cem\u003ein vitro\u003c/em\u003e measured activity involves only a partial reaction, the first steps of Cys desulfurase reaction, employing DTT as a non-physiological reducing agent. In contrast, in the cell, the Fe-S cluster formation to sustain Fe-S dependent enzymes involves several steps, including the persulfide transfer to ISCU2, the electron delivery (reduction) by FDX2, the iron-sulfur cluster assembly on ISCU2, and the cluster transferring to target subunits and enzymes.\u003c/p\u003e \u003cp\u003eThe dynamic behavior of the L-Cys NFS1 desulfurase supercomplex was further characterized last year by including the electron donor for cluster assembly Ferredoxin 2 (FDX2) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recently, it was determined that this protein has a multifaceted binding mode (two steps involving the C-terminal partially folded stretch of FDX2) to the supercomplex [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Moreover, it was demonstrated that FDX2 binds to the same supercomplex region as FXN. This indicates that precise exchange dynamics are essential for accurate function. Changes in FXN or FDX2 affinity by the supercomplex could significantly impact the kinetics of the catalytic cycle. The interaction of FXN with the NB, making subtle clashes of the NB and NFS1 structures, might have unexpected effects on function, suggesting that the reaction studied \u003cem\u003ein vitro\u003c/em\u003e, the L-Cys desulfurization in the presence of DTT, may show a different profile compared to the complete reaction occurring in the cell, involving the Fe-S cluster formation, which depends on FDX2/FXN dynamics.\u003c/p\u003e \u003cp\u003eThis work suggests the possibility of rescuing pathogenic FXN variants characterized by lower conformational stability and an increased tendency to be degraded compared to wild-type FXN and opens the door to a general strategy for intervening in human mitochondrial biochemistry.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacyl carrier protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecircular dichroism\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCTR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eC-terminal region\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFe-S\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eiron-sulfur\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFriedreich\u0026rsquo;s Ataxia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFXN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efrataxin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edithiothreitol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-performance liquid chromatography\u0026lrm;\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFast protein liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eISCU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eiron-sulfur cluster assembly enzyme\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eISD11\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNFS1 interacting protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNFS1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial L-cysteine desulfurase enzyme\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear magnetic resonance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolyacrylamide gel electrophoresis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein Data Bank\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esodium dodecyl sulfate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esize exclusion chromatography.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by Universidad de Buenos Aires UBACyT20020190100338BA, CONICET, and Friedreich´s Ataxia Research Alliance (FARA, grant 2023-2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM.F.P. and J.S. performed experiments, made the analysis of the experiments and structures, and wrote the paper.\u003c/p\u003e\n\u003cp\u003eN.B. F. made co-immunoprecipitations, western blots, and analysis of the results\u003c/p\u003e\n\u003cp\u003eA. G., R. M., and J. A. H. D. performed the crystallization, determined the X-ray structure, analyzed the structures, and wrote the paper.\u003c/p\u003e\n\u003cp\u003eJ.G., A.G., and N.A.R. purified the NBs\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM. N. purified FXN and the NFS1/ACP-ISD11 and ISCU proteins.\u003c/p\u003e\n\u003cp\u003eA.V. \u0026nbsp;acquisition and processing of fluorescence microscopy image.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH.G.G. measured ACO and SDH enzymic activities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM. A. acquired 2D NMR spectra\u003c/p\u003e\n\u003cp\u003eL.I.I, M.P.P, G.V.P, and M.B. made the library of nanobodies and the selection by phage display\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFan, X., et al., \u003cem\u003eIron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.\u003c/em\u003e J Biol Chem, 2022. \u003cstrong\u003e298\u003c/strong\u003e(7): p. 102094.\u003c/li\u003e\n \u003cli\u003ePandey, A.K., et al., \u003cem\u003eMitochondria export iron-sulfur and sulfur intermediates to the cytoplasm for iron-sulfur cluster assembly and tRNA thiolation in yeast.\u003c/em\u003e J Biol Chem, 2019. \u003cstrong\u003e294\u003c/strong\u003e(24): p. 9489-9502.\u003c/li\u003e\n \u003cli\u003ePaul, V.D. and R. 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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.\u003c/p\u003e \u003cp\u003eIn 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 (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e= 1\u0026ndash;30 nM) and stabilized the pathogenic FXN variant G130V by increasing its Tm by 15\u0026deg;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.\u003c/p\u003e \u003cp\u003eThe 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.\u003c/p\u003e","manuscriptTitle":"Nanobodies as Novel Tools to Modulate Human Frataxin Stability and Function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-12 06:33:06","doi":"10.21203/rs.3.rs-6122246/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7c8501a7-8c4a-4ae2-972d-854ba11bbdad","owner":[],"postedDate":"March 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45472251,"name":"Biological sciences/Biophysics/Molecular biophysics/Supramolecular assembly"},{"id":45472252,"name":"Biological sciences/Biotechnology/Molecular engineering/Protein design"},{"id":45472253,"name":"Biological sciences/Structural biology/X-ray crystallography"}],"tags":[],"updatedAt":"2026-02-07T08:07:22+00:00","versionOfRecord":{"articleIdentity":"rs-6122246","link":"https://doi.org/10.1038/s42003-025-09458-x","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2026-01-03 05:00:00","publishedOnDateReadable":"January 3rd, 2026"},"versionCreatedAt":"2025-03-12 06:33:06","video":"","vorDoi":"10.1038/s42003-025-09458-x","vorDoiUrl":"https://doi.org/10.1038/s42003-025-09458-x","workflowStages":[]},"version":"v1","identity":"rs-6122246","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6122246","identity":"rs-6122246","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}