Molecular View of an Electron Transfer Process Essential for Iron–Sulfur Protein Biogenesis

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

Lucia Bancia,b,1, Ivano Bertinia,2, Vito Calderonea,b, Simone Cioﬁ-Baffonia,b, Andrea Giachettia, Deepa Jaiswala, Maciej Mikolajczyka, Mario Picciolia,b, and Julia Winkelmanna

aMagnetic Resonance Center and bDepartment of Chemistry, University of Florence, 50019 Sesto Fiorentino, Florence, Italy

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved March 22, 2013 (received for review February 5, 2013)

Biogenesis of iron–sulfur cluster proteins is a highly regulated pro- oxidoreductase 1 (Ndor1) and anamorsin, form a stable complex in cess that requires complex protein machineries. In the cytosolic vivo that can functionally replace the Tah18-Dre2 complex in yeast iron–sulfur protein assembly machinery, two human key proteins— cells (13, 15). Similar to Dre2 but with significant differences in the NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsin— number of residues, anamorsin contains two domains (an N-ter- form a stable complex in vivo that was proposed to provide electrons minal domain of 172 residues and a C-terminal domain of 90 res- for assembling cytosolic iron–sulfur cluster proteins. The Ndor1–ana- idues, named cytokine-induced apoptosis inhibitor 1 (CIAPIN1) morsin interaction was also suggested to be implicated in the regu- hereafter, which contains two highly conserved cysteine rich motifs, lation of cell survival/death mechanisms. In the present work we CX8CX2CXC and CX2CX7CX2C) connected by a linker of 51 unravel the molecular basis of recognition between Ndor1 and ana- residues (18). Recently, we reported the solution structure of the morsin and of the electron transfer process. This is based on the well-folded N-terminal domain of anamorsin [Protein Data Bank structural characterization of the two partner proteins, the investiga- (PDB) ID: 2LD4] and showed that the CIAPIN1 domain of ana- tion of the electron transfer process, and the identification of those morsin, at variance with Dre2, does not bind a [4Fe-4S] cluster but protein regions involved in complex formation and those involved in binds a [2Fe-2S] cluster through the CX8CX2CXC motif of CIA- electron transfer. We found that an unstructured region of anamor- PIN1 (18). Recent EPR data also support the presence of only the [2Fe-2S] cluster(s) in Dre2 (19). sin is essential for the formation of a specific and stable protein com- Like yeast Tah18, human Ndor1 belongs to the family of diflavin plex with Ndor1, whereas the C-terminal region of anamorsin, reductases and consists of two domains: the first binds FMN containing the [2Fe-2S] redox center, transiently interacts through (FMN-binding domain, hereafter), and the second binds FAD and complementary charged residues with the FMN-binding site region NADPH (FAD-binding domain, hereafter). On the basis of the of Ndor1 to perform electron transfer. Our results propose a molecu- well-known electron transfer mechanism occurring in diflavin re- lar model of the electron transfer process that is crucial for under- ductase enzymes (14) (i.e., electrons are transferred from NADPH standing the functional role of this interaction in human cells. to FAD and then to FMN, which serves as a donor for one-electron terminal acceptors), the FMN-binding domain is the part of Ndor1 Fe/S protein maturation | CIAPIN1 domain | diflavin reductase directly interacting with the terminal electron acceptor anamorsin. On this basis, the FMN-binding domain of Ndor1 has been used ron–sulfur clusters (ISCs) are ancient inorganic cofactors that are within this study. Icrucial for many protein functions in eukaryotic and bacterial cells In the present work we unravel the molecular basis of the rec- (1–3). The clusters are composed of inorganic sulfide and ferric/ ognition and of the electron transfer process between Ndor1 and ferrous iron atoms, the latter being preferentially coordinated by anamorsin. This is based on the structural characterization of the cysteinyl residues (4–6). Because inorganic sulfide and ferrous/ferric FMN-binding domain of Ndor1 and of the C-terminal region iron atoms are toxic in vivo, biosynthesis of ISC proteins within cells (linker and CIAPIN1 domain) of anamorsin containing the [2Fe- is a highly regulated process that requires complex protein ma- 2S] cluster in the oxidized state ([2Fe-2S]-anamorsin, hereafter) chineries for the mobilization of Fe and S atoms from appropriate and on the identification of the protein regions between [2Fe-2S]- sources, for their assembly into ISC forms and their final delivery to anamorsin and the FMN-binding domain of Ndor1 responsible for the recipient proteins (7–9). Three distinct protein machineries are the formation of their stable complex and of those regions inter- operative and essential in the (nonplant) eukaryotic cells for the acting in the electron transfer process. The molecular model of the biogenesis of ISC proteins: (i) the ISC assembly machinery in the electron transfer process proposed here provides significant in- mitochondrial matrix, (ii) the ISC export machinery located in formation on the functional processes in which the anamorsin– the mitochondrial intermembrane space, and (iii) the cytosolic Ndor1 interaction has been implicated, i.e., the assembly of ISCs iron–sulfur protein assembly (CIA) machinery. (13) and diferric (17) proteins and the regulation of cell survival/ The CIA machinery comprises several proteins (10, 11), among death mechanisms (15, 16). which one named Dre2 has been recently identified in yeast (12). The C-terminal domain of Dre2 (residues 228–348) is able to bind Results two ISCs, a [2Fe-2S] and a [4Fe-4S] (12, 13). The [2Fe-2S] cluster fl Structural Characterization of the FMN-Binding Domain of Ndor1. of Dre2 receives electrons from a cytosolic di avin reductase, The crystal structure of the FMN-binding domain of Ndor1 termed Tah18 (13), which contains a FAD and a FMN prosthetic group, respectively, bound in two distinct structural domains, to accept electrons from NADPH (14). Dre2 and Tah18 are protein partners forming a stable complex in vivo (13, 15). The C-terminal Author contributions: L.B., I.B., and S.C.-B. designed research; V.C., S.C.-B., A.G., D.J., M.M., – M.P., and J.W. performed research; V.C., S.C.-B., A.G., M.M., M.P., and J.W. analyzed data; region of Dre2 (residues 173 348) is fundamental to form, both in and L.B., S.C.-B., M.M., M.P., and J.W. wrote the paper. vivo and in vitro, the stable complex with Tah18 (16). The Tah18- fl Dre2 interaction is essential for yeast viability and it has been The authors declare no con ict of interest. implicated in cellular death regulation mechanisms (16). This This article is a PNAS Direct Submission. complex was proposed to provide electrons necessary for the CIA Data deposition: The atomic coordinates and structure factors have been deposited in the machinery (13) and for assembling the diferric tyrosyl radical co- Protein Data Bank, www.pdb.org (PDB ID code 4H2D). factor of ribonucleotide reductase, Rnr2 (17). However, the mo- 1To whom correspondence should be addressed. E-mail: [email protected]fi.it. lecular basis of these processes has not been elucidated. The 2Deceased July 7, 2012. electron transfer process observed in yeast is possibly conserved in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. humans as the functional orthologs, NADPH-dependent diflavin 1073/pnas.1302378110/-/DCSupplemental.

7136–7141 | PNAS | April 30, 2013 | vol. 110 | no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1302378110 Downloaded by guest on September 27, 2021 + (FMN-Ndor1 hereafter) shows the classical fold of FMN-binding [2Fe-2S]2 cluster (18) was structurally characterized through domains of diflavin reductases (20, 21) and consists of a wound solution NMR. The linker does not show any specific secondary α-β-α fold with five parallel β-strands (the fifth strand divided in structural element with the exception of the short hydrophobic two short β-strands with a gap of four residues) in the core of the stretch 192–195 showing some α-helical secondary structure pro- molecule flanked by two helices on one side (α1andα5) and three pensity (Fig. S1). The simultaneous occurrence of paramagnetic (α2, α3, and α4) on the other (Fig. 1A and Table S1). This structural effects on resonance linewidths, of poor chemical shifts dispersion of organization is fully maintained in solution (SI Materials and the NMR signals, and of extensive resonance overlap made the Methods). A search for structurally related proteins using the Dali structural characterization of the CIAPIN1 domain in full-length server identifies the FMN-binding domain of the diflavin reductase anamorsin essentially impossible. Therefore, we cloned and pro- NADPH-cytochrome P450 reductase (FMN-CytP450r hereafter, duced it as an independent domain in a construct reported as iso- PDB ID 1B1C, Z-score of 23.9, rmsd 1.5 Å) as the closest structural form 2 in the UniProt database (residues 205–312, CIAPIN1-single homolog in the human proteome. With the exception of the ab- hereafter). The electrospray ionization (ESI)-MS, UV/visible, and sence, in FMN-Ndor1, of a N-terminal helix, which is spatially close EPR spectra of CIAPIN1-single showed the same spectroscopic to helix α2 in FMN-CytP450r, the α-helices and β-strands involve features for the [2Fe-2S] cluster as those in [2Fe-2S]-anamorsin (Fig. a similar number of residues (Fig. 1B). On the contrary, the loops S2 and SI Materials and Methods), indicating that CIAPIN1-single show more variability, in the main affecting two spatially close loops maintains the same coordination and electronic properties of the connecting helix α2 and strand β3 and helix α3 and strand β4, re- [2Fe-2S] cluster bound in the full-length protein. The circular di- SI spectively (Fig. 1B). These loops in FMN-Ndor1 are, respectively, chroism spectrum of [2Fe-2S]-CIAPIN1-single (Fig. S3 and Materials and Methods shorter and longer than those in FMN-CytP450r, thereby making ) shows that the protein adopts conformations α α typically found in unfolded proteins with a little α-helical content. In helix 2 and helix 3 shorter by two residues in FMN-Ndor1 (Fig. 1 15 1B). Although structurally very similar, FMN-Ndor1 and FMN- the H- N heteronuclear single quantum coherence (HSQC) CytP450r are quite different in terms of charged residues and spectrum of the [2Fe-2S]-CIAPIN1-single (Fig. S3) cross-peaks are charge surface distribution. The majority of these differences are highly crowded in the spectral region between 8 and 8.5 ppm and α α well superimposable to the corresponding cross-peaks of the located in helices 2and 3, which are close to each other on the 1 15 same face of the β-sheet. They are both negatively charged in FMN- H- N HSQC spectrum of [2Fe-2S]-anamorsin. These data in- CytP450r whereas they are, respectively, neutral and positively dicate that the CIAPIN1 domain also in the full-length anamorsin is largely unstructured with no large tertiary structure organi-

C CHEMISTRY charged in FMN-Ndor1 (Fig. 1 ). A negative electrostatic potential fi around the FMN-binding site is still maintained in FMN-Ndor1, zation and that there are no speci c interactions of the CIA- even if to a lesser extent than that in FMN-CytP450r (Fig. 1C). PIN1 domain with the linker and the N-terminal domain. Close to zero or negative 15N{1H}-NOE values, typical of unstructured Structural Characterization of the C-Terminal Region of [2Fe-2S]- proteins, as well as R2/R1 ratios lower than expected for a globular Anamorsin. The C-terminal region of [2Fe-2S]-anamorsin, protein of the CIAPIN1-single size, are found along the amino – acid sequence of [2Fe-2S]-CIAPIN1-single, with the exception of which includes the linker (residues 173 223) and the CIAPIN1 15 domain (residues 224–312) coordinating an oxidized, paramagnetic some residues in the CX2CX7CX2C motif that have positive N {1H}-NOE values, indicating their tendency to adopt a partially defined conformation, but still not characterized by a stable 3D structure (Fig. S3). It can be concluded from the available A B chemical shift values that residues 254–264, a stretch that connects the two cysteine motifs, residues 229–235, which precede the metal- binding motif, and some residues located in the CX CX CX C 5 3 2 7 2 4 motif, have a propensity to be in an α-helical conformation (Fig. 3 S3), whereas all of the other residues have essentially a random coil conformation, in full agreement with the low α-helical content 1 4 observed in the circular dichroism spectrum. Backbone NHs affected by fast, paramagnetic relaxation prop- 1 15 2 erties were identified through a H- N inversion recovery filtered 3 2 HSQC experiment observed in antiphase (IR-HSQC-AP) specifically designed to identify scalar couplings between fast relaxing 1H Negatively resonances. Thirteen backbone NH resonances, 10 of which were C charged 1 15 area lost in standard diamagnetic H- N HSQC experiments, were detected with this experiment (Fig. 2A). 1H- and 13C-detected triple resonance experiments tailored to assign fast relaxing 1H, 15 13 Neutral, Positively N, and C resonances were also performed (22–25). An as- hydrophobic charged signment can be proposed for 7 of the 10 NH resonances observed area area 1 15 3 only in the H- N IR-HSQC-AP experiment (Fig. 2A) and for 7 of 2 the 11 Cα and CO resonances observed only in the 13C-detected paramagnetic experiments. These fast relaxing, assigned residues Fig. 1. Structure of the FMN-binding domain of Ndor1. (A) Ribbon repre- are located in the regions encompassing both CX8CX2CXC and 1 13 sentation of the crystal structure of the FMN-binding domain of Ndor1. FMN CX2CX7CX2Cmotifs.Their Hand C longitudinal relaxation rates is shown as beige spheres. (B) Superimposition of the crystal structures of the were measured, providing precious information on the relative po- FMN-binding domains of Ndor1 (green, FMN in beige spheres) and of the sition of these residues with respect to the [2Fe-2S] cluster. A human NADPH-cytochrome P450 reductase (PDB ID: 1B1C) (red, FMN in or- structural model of [2Fe-2S]-CIAPIN1-single was calculated using ange spheres). The regions with structural variations between the two pro- 20 distance restraints that define the [2Fe-2S] cluster binding to the teins are shown in gray. (C) Molecular surface of the FMN-binding domain of protein, 17 paramagnetic-based distance restraints, obtained from Ndor1 (Left) and of the human NADPH-cytochrome P450 reductase (Right) 1 13 α colored according to their electrostatic potential. The views are equivalent in longitudinal HN and C relaxation rates, and 51 dihedral angle SI Materials and Methods terms of the orientation of the protein backbone. The colors of the molecular restraints (see for details). This structural surface indicate positive (blue), neutral (white), and negative (red) electro- model was then subjected to an unrestrained molecular dy- static potentials. The FMN molecule is shown as orange spheres. namics (MD) simulation of 100 ns to explore possible, further

Banci et al. PNAS | April 30, 2013 | vol. 110 | no. 18 | 7137 Downloaded by guest on September 27, 2021 structurally different from FMN-CytP450r (Fig. 4A). Smaller 15N ABG280 spectral changes are also observed for several residues in the four A234 loops surrounding the FMN moiety (Fig. 4A). The residues directly involved in protein–protein recognition, based on their G292 S272 T250 G299 large chemical shift changes as well as their high solvent acces-

D281 R243 sibility (relative solvent accessibility above 50%), identify three A282 main areas characterized by negatively charged, positively N248 C K241 charged, and hydrophobic residues, respectively (Figs. 1 and A245 4A), with the negatively charged area surrounding only the FMN- 1H binding site. When analyzing the interaction from the anamorsin side, it Fig. 2. Structural characterization of [2Fe-2S]-CIAPIN1-single. (A) 1H-15NIR- appears that chemical shift changes in the 1H-15N HSQC maps of 1 HSQC-AP spectrum, optimized to detect fast relaxing H resonances, showing oxidized, 15N-labeled [2Fe-2S]-anamorsin involve only signals 13 backbone NH resonances of the [2Fe-2S]-CIAPIN1-single, 10 of which present in the crowded 1H(NH) region between 8 and 8.5 ppm, (in black) were completely lost in the standard diamagnetic 1H-15NHSQC experiment (the three residues also detected in the diamagnetic 1H-15N whereas all of the resonances of the folded N-terminal domain HSQC are shown in gray. (B) Representative model of the CIAPIN1 domain remain essentially unaffected (Fig. S5). This behavior indicates of anamorsin derived from a molecular dynamics simulation of 100 ns. The that the unstructured C-terminal region of anamorsin interacts

[2Fe-2S] cluster bound to the CX8CX2CXC motif is shown; the four cysteines with oxidized FMN-Ndor1, whereas the N-terminal, well-struc- coordinating the [2Fe-2S] cluster and the four cysteines of the CX2CX7CX2C tured domain of anamorsin is not involved in protein–protein motif are shown. recognition. Accordingly, no chemical shift perturbations were observed when oxidized, 15N-labeled FMN-Ndor1 was titrated with the N-terminal domain of [2Fe-2S]-anamorsin produced as an conformations. The MD simulation showed that the [2Fe-2S]- independent domain (Fig. S4). This result is consistent with the cluster–bound CX CX CXC region and the CX CX CX C region 8 2 2 7 2 absence of any interaction between the N-terminal domain of Dre2 sample a restricted range of conformations that are close in space and Tah18, as monitored both in vitro and in vivo (16). When the and with an α-helix stably formed between them (residues 254–268, 13 15 B anamorsin/FMN-Ndor1 interaction was monitored on a C, N- Fig. 2 ), in full agreement with the backbone diamagnetic NMR labeled oxidized [2Fe-2S]-anamorsin sample by 13C-direct de- chemical shifts (Fig. S3) and the paramagnetic longitudinal re- tection CON, CACO, and CBCACO NMR experiments (Fig. S5), laxation rates. which experience increased spectral resolution for unstructured proteins (26), the spectral changes are located in the region pre- Molecular Recognition and Electron Transfer Between the FMN- ceding the CX CX CXC motif (residues 185–223), encompassing Binding Domain of Ndor1 and [2Fe-2S]-Anamorsin. When 15N-labeled 8 2 the last 38 residues of the linker. This region can be partitioned in oxidized FMN-Ndor1 is titrated with unlabeled oxidized [2Fe-2S]- fi 1 15 two main areas characterized by speci c amino acid content: anamorsin, the H- N HSQC spectra are drastically affected, a highly hydrophobic area (residues 188–202) and a negatively indicating that the two proteins interact. The majority of the charged area (residues 204–223) (Fig. S6). These two areas match, NH cross-peaks broaden beyond detection upon protein addition, respectively, the hydrophobic and positively charged areas of with the exception of the last 15 residues of the C-terminal un- FMN-Ndor1 involved in protein recognition, which are not directly structured tail of FMN-Ndor1 that remain unaffected in the C 1 15 in contact with the FMN cofactor (Fig. 1 ). Such interactions are H- NHSQCmaps(Fig. S4), indicating that they are not involved thus those responsible for the formation of a specific and stable in the interaction and still reorient faster than the overall protein protein complex between Ndor1 and anamorsin. tumbling rate. The majority of the NH signals can be recovered Oxidized [2Fe-2S]-CIAPIN1-single interacts also with oxidized in a transverse relaxation optimized spectroscopy (TROSY)-type FMN-Ndor1, but on a fast exchange regime on the NMR time- 1 15 H- N HSQC experiment, which can detect signals with broader scale (Fig. 3B). The chemical shift changes on FMN-Ndor1 are linewidths (Fig. 3A). These NMR data indicate the formation of smaller than those detected when the complex is formed with a tight, large molecular mass complex, which is in slow exchange, [2Fe-2S]-anamorsin and involve a lower number of residues (Figs. on the NMR timescale, with the isolated proteins. The chemical 3 and 4). Still, on the FMN-Ndor1 structure the interacting res- shift variations observed in the 1H-15N HSQC spectra of the idues are localized in an area that is part of the interacting surface complex vs. FMN-Ndor1, mapped on the structure of the FMN- of the complex between [2Fe-2S]-anamorsin and FMN-Ndor1 binding domain, indicated a well defined protein–protein recog- (Fig. 4B). These results indicate that a specific protein–protein nition region (Fig. 4A). Specifically, the major spectral changes interaction still occurs but the two proteins do not form a stable are observed on the spatially close helices α2, α3, and α4andon complex (Fig. 3). Therefore, the hydrophobic interacting residues the two loops following helices α2andα3, described above as of the linker (188–202, Fig. S6), which are absent in the [2Fe-2S]-

Fig. 3. Protein–protein interaction between the FMN-binding domain of Ndor1 and [2Fe-2S]-anamorsin as characterized by NMR. (A) TROSY 1H-15N HSQC at 308 K, acquired at 900 MHz, of the 15N- labeled oxidized FMN domain (construct 1–174 aa) before (red) and after (black) the addition of 1 eq of unlabeled oxidized [2Fe-2S]-anamorsin. (B) Overlay of 1H-15N HSQC spectra of 15N-labeled oxidized FMN-binding domain at 298 K, acquired at 800 MHz, before (black) and after (red) addition of 1 eq of [2Fe-2S]-CIAPIN1-single. (Inset) Chemical shift variations of selected FMN-binding domain residues upon addition of increasing amounts of [2Fe-2S]- CIAPIN1-single (0%, 50%, and 100%).

7138 | www.pnas.org/cgi/doi/10.1073/pnas.1302378110 Banci et al. Downloaded by guest on September 27, 2021 addition of 1 eq of unlabeled, oxidized FMN-Ndor1. With the exception of one signal, all of the others show Δavg(HN) values [i.e.,((ΔH)2 + (ΔN/5)2)/2)1/2,whereΔHandΔN are chemical shift differences for 1Hand15N, respectively] with less than 0.03 ppm variations (Fig. S5), despite the fact that the two proteins form the tight, large molecular mass complex. The occurrence of small chemical shift variations suggests the presence of interactions with no specific orientation at the redox centers. Rather, there is a dynamic ensemble of orientations governed predominantly by long- range electrostatic forces as already observed in other electron transfer protein complexes (28, 29). This is in agreement with the presence of a positively charged region nearby the [2Fe-2S] cluster (Fig. S6) and a negatively charged region surrounding the FMN- binding site (Fig. 1C). Electron transfer over a sufficiently short distance is still possible in multiple orientations and the re- Fig. 4. Mapping the binding interface of the FMN-binding domain of quirement to form a specific interaction is less stringent (28). This is Ndor1 interacting with [2Fe-2S]-anamorsin. (A and B) Ribbon representa- especially true in the present case in which the specificity in pro- tions of the FMN-binding domain showing as spheres backbone NHs of the – residues experiencing chemical shift variations upon interaction with (A) tein protein recognition is already guaranteed by intermolecular [2Fe-2S]-anamorsin and (B) [2Fe-2S]-CIAPIN1-single. Large blue spheres, res- contacts involving hydrophobic and complementary charged resi- idues showing large chemical shift changes and characterized by relative dues far from the [2Fe-2S] cluster and FMN regions. solvent accessibility above 50%; small blue spheres, residues showing large As the interaction between anamorsin and FMN-Ndor1 still chemical shift changes but featuring relative solvent accessibility lower than occurs also between apo-anamorsin and oxidized FMN-Ndor1 and 50%; large cyan spheres, residues showing small chemical shift changes with between oxidized [2Fe-2S]-anamorsin and fully reduced FMNH2- concomitant broadening effects and with relative solvent accessibility above Ndor1, as monitored through NMR experiments (Fig. S8), it results 50%; small cyan spheres, residues showing small chemical shift changes with that the redox state of the FMN moiety as well as the presence or concomitant broadening effects and with relative solvent accessibility lower not of the [2Fe-2S] cluster does not affect the formation of the than 50%. Positively charged (gray), negatively charged (red), and hydro- protein complex. This behavior suggests that the protein–protein phobic (yellow) side chains which are highly solvent exposed in the inter- CHEMISTRY acting region are indicated. interaction interface is not affected by the redox centers and is fully consistent with a protein–protein recognition process determined by residues far from the redox centers. The same behavior was CIAPIN1-single construct, are essential to determine an effective reported for the in vitro interaction of the yeast homologs, where recognition of anamorsin. apo-Dre2 showed stable interaction with Tah18 (16). To detect electron transfer between the FMN-binding domain of Ndor1 and [2Fe-2S]-anamorsin, we used UV-visible spectros- Discussion copy. FMN-Ndor1 can be reduced anaerobically to form the In this work, we defined the molecular basis for the recognition • neutral blue semiquinone state (FMNH ) and the fully reduced between the diflavin reductase Ndor1 and the iron–sulfur protein state (FMNH2) of FMN by addition of 0.50 and 1.0 eq of anamorsin and for the electron transfer process between them. dithionite, respectively (Fig. S7 and SI Materials and Methods for We showed that the two proteins form a stable complex where details). FMN-Ndor1 in either of the reduced states was titrated one electron is transferred from the hydroquinone state of the with [2Fe-2S]-anamorsin or [2Fe-2S]-CIAPIN1-single in their ox- FMN moiety of Ndor1 to the oxidized [2Fe-2S] cluster of ana- fi – idized states and the reactions were followed by UV-visible spec- morsin. The stable complex is achieved thanks to speci c protein • troscopy. When FMNH was mixed with [2Fe-2S]-anamorsin or protein recognition between a completely unstructured region of – with [2Fe-2S]-CIAPIN1-single, no changes were observed in the anamorsin (residues 185 223), which is part of the linker sepa- UV-visible spectra, indicating that no electron transfer occurs rating the N-terminal domain from the C-terminal CIAPIN1 • α – between the FMNH moiety and the [2Fe-2S] redox center. On the domain, and a -helical containing face of the FMN-binding domain of Ndor1. This molecular recognition is specifically gov- contrary, when FMNH was mixed with [2Fe-2S]-anamorsin or 2 erned by hydrophobic and complementary charged interacting with [2Fe-2S]-CIAPIN1-single, electrons were stoichiometrically residues. In particular, the hydrophobic interactions are essential transferred from FMN to [2Fe-2S], producing the reduced state of • to ensure the formation of a stable complex, the electrostatic the [2Fe-2S] cluster and the semiquinone FMNH species. Indeed, interactions are determining only a transient complex between the absorbance at 432 nm, i.e., the isosbestic point of the FMNH2 • anamorsin and the FMN-binding domain of Ndor1, and the and FMNH species, decreases over time, indicating the reduction N-terminal domain of anamorsin is not involved in the recogni- of the [2Fe-2S] cluster because the latter absorbs at this wavelength tion process. The formation of the stable complex is independent only in its oxidized state (Fig. S7); and the formation of the • of the presence of the [2Fe-2S] center as well as of the redox state FMNH state is monitored by the increase of the absorbance at 650 • of the FMN moiety, indicating that the two protein partners do nm that is due only to the presence of the FMNH species (Fig. S7). interact permanently and no dissociation occurs along the elec- The rate of the [2Fe-2S] cluster reduction is of the same order of tron transfer process. The two redox centers, on the other hand, magnitude in [2Fe-2S]-anamorsin and in [2Fe-2S]-CIAPIN1-sin- transiently interact via electrostatic interactions between a nega- gle (Fig. S7). Because the midpoint reduction potentials of the tively charged region surrounding the FMN moiety and a positively oxidized/semiquinone and semiquinone/dihydroquinone couples charged region surrounding the [2Fe-2S] cluster. All these data present in the FMN-binding domain of Ndor1 are −146 mV and lead to a molecular model for the protein–protein recognition and −305 mV (27), respectively, the reduction potentials of the [2Fe- a + + for the electron transfer process according to which ( ) the two 2S] 2/[2Fe-2S] 1 cluster center in anamorsin have to be in between proteins form a stable complex through specific interactions in- these two values. volving regions that are not in direct contact with the redox To specifically monitor the interaction between the two redox cofactors; (b) the areas surrounding the FMN and [2Fe-2S] redox centers ([2Fe-2S] cluster and FMN) involved in the electron moieties transiently and weakly interact with each other, ex- transfer process, paramagnetic 1H-15NIR-HSQC-APmapsofox- changing one electron; and (c) the unstructured region comprising idized [2Fe-2S]-anamorsin were collected before and after the residues 185–223 is essential for the complex formation and can

Banci et al. PNAS | April 30, 2013 | vol. 110 | no. 18 | 7139 Downloaded by guest on September 27, 2021 Closed electrons from NADPH to FMN through the mediation of FAD. conformation Indeed, when the region of the FMN-binding domain of Ndor1, of Ndor1 which stably interacts with anamorsin, is mapped on a structural model of entire Ndor1 (obtained from the crystal structure of the homologous human cytochrome P450 reductase in the closed conformation where the FMN and FAD moieties are in close CIAPIN1 domain contact to each other, thus ensuring an efficient interflavin electron of anamorsin transfer), this region is still largely solvent exposed and therefore N-terminal still able to interact with anamorsin (Fig. 5). Consistent with this, domain of Linker of anamorsin anamorsin coimmunoprecipitation data showed that anamorsin and Ndor1 NADPH form a stable complex in vivo (13). The transient interaction observed for the protein regions containing the two redox cofactors FMN and [2Fe-2S] also fits well in the overall electron transfer fl fl Closed process of di avin reductases. For di avin reductase enzymes, it is conformation well established that a conformational change from the closed of Ndor1 conformation to a more open one is required to promote the final electron transfer from FMNH2 to its substrate (30). Otherwise, the reduced FMN would have too restricted solvent accessibility in the closed conformation to efficiently deliver electrons to the sub- strates (30–32). It has been also found that the open conformational state is significantly populated in the NADPH-bound, reduced state of diflavin reductases (31, 33, 34). At variance with the stable interaction between the FMN-binding domain of Ndor1 and the unstructured region of anamorsin, the transient interaction observed in the regions containing the FMN and [2Fe-2S] cofactors can be easily regulated upon the occurrence of the conformational closed-to-open equilibrium of the FAD/FMN domains of Open Ndor1. Indeed, the FMN-[2Fe-2S] interaction could be favored conformation of Ndor1 when the interacting area of Ndor1 surrounding the FMN moiety is solvent exposed in the open conformation and unfavored in the closed conformation, which does not expose this interacting area (Fig. 5). An electron transfer process coupled with tightly regulated conformational rearrangements can therefore be proposed to oc- cur in the anamorsin–Ndor1 complex (Fig. 5): (i) Anamorsin is stably bound to both closed and open conformations of Ndor1 due to a specific recognition between an unstructured region of anamorsin and a region of the FMN-binding domain that is solvent exposed in both open and closed conformations; (ii)uponNADPH fi Open binding by Ndor1, electrons can ef ciently be transferred within the conformation closed conformation of Ndor1 to produce the hydroquinone state of Ndor1 of FMN; (iii) NADPH binding and interflavin electron transfer within Ndor1 significantly populate the open conformation, which allows the formation of the transient interaction between the FMN and the [2Fe-2S] cluster regions; and (iv) the latter interaction allows efficient transfer of one electron from the hydroquinone state of FMN to the [2Fe-2S] cluster. Our study lays out the molecular basis for the comprehension of the two functional processes in which the anamorsin–Ndor1 complex is implicated, i.e., assembly of Fe/S proteins and regulation of Fig. 5. Model of the electron transfer process between Ndor1 and anamorsin. Anamorsin (in pink) can be tightly bound to both closed and open confor- cell survival/death mechanisms. From our data we can propose that mational states of Ndor1 (in gray) due to the specific recognition between an the electron transfer process responsible for the assembly of ISC unstructured region of anamorsin and a region of the FMN-binding domain (13)anddiferric(17)proteinsoccurswithinastablecomplexwith- that is solvent exposed in both open and closed conformations (in yellow). The out the dissociation of the two protein partners but just through the N-terminal domain of anamorsin is not involved in the protein–protein rec- modulation of the interactions of the domains involved in the ognition process. Upon NADPH (in blue) binding, electrons can efficiently be electron transfer process. Although the molecular targets of the transferred in the closed conformation of Ndor1 to FAD (in red) and then to electron transfer flow generated by this protein–protein complex are FMN (in green) to produce the hydroquinone state of FMN. Interflavin elec- not yet defined, suggestions for the targets of the electron flow in- fi tron transfer in Ndor1 signi cantly populates the open conformation, which clude the conversion of the sulfur of cysteine (formally S0)tothe exposes to the solvent the FMN moiety and allows the formation of a transient fi 2− interaction with the [2Fe-2S] cluster region of anamorsin located in the CIA- sul de (S ) present in Fe/S clusters and/or the reductive coupling of PIN1 domain. The latter interaction allows an efficient transfer of one electron two [2Fe-2S] clusters to form a [4Fe-4S] cluster (13). This non- from the hydroquinone state of FMN to the [2Fe-2S] cluster (in black). dissociative electron transfer process might also rationalize how anamorsin regulates cell survival/death mechanisms in human cells (35) and why a stable Dre2-Tah18 interaction is essential for yeast contribute in positioning the CIAPIN1 domain containing the viability (16). Indeed, the disruption of the stable interaction be- [2Fe-2S] cluster in those orientations that can efficiently receive tween anamorsin and Ndor1 might provoke the interruption of the the electron(s) from the hydroquinone state of the FMN redox electron flow between the two proteins within the cell and, as center. This molecular model nicely fits with the overall electron a result of that, its essential function for cellular survival is abol- transfer chain occurring in diflavin reductases, which deliver ished and consequently cell death mechanisms might be activated.

7140 | www.pnas.org/cgi/doi/10.1073/pnas.1302378110 Banci et al. Downloaded by guest on September 27, 2021 Materials and Methods 2,048 scans per fid were acquired, using inversion recovery, inept transfer, and Protein Production. A detailed procedure of cloning and protein production recycle delays of 50 ms, 0.83 ms, and 55 ms, respectively. Fast spin relaxation of fi of the FMN-binding domain of Ndor1 is reported in SI Materials and paramagnetic signals, however, decreases dramatically the ef ciency of co- Methods. The DNA encoding CIAPIN1-single of anamorsin (205–312 aa) was herence transfer and prevents resonance assignment via the standard proto- 1 13 amplified by PCR from the Gateway pEntr-TEV-d-Topo vector containing cols that rely on H- or C-direct–detected triple resonance experiments. NMR full-length anamorsin and inserted into the same vector. Full-length ana- experiments tailored to assign resonances affected by the presence of a para- morsin, CIAPIN1-single, and the N-terminal domain of anamorsin were magnetic center were therefore performed on a Bruker AVANCE 500 or on expressed, purified, and chemically reconstituted following a previously a Bruker AVANCE 700, specifically modifying HNCA, HNCO, CBCACONH, and reported procedure (18). 13C-detected CON, CC-COSY, CACO, IR-CACO-AP, and CBCACO pulse sequences (see SI Materials and Methods for details). All NMR data were processed using X-Ray Crystallography. Crystals of the FMN-binding domain of Ndor1 (1–161 aa) the Topspin software package and were analyzed with the program CARA were obtained using the vapor diffusion technique at 20 °C from 1.3-mM (36). Secondary structure analysis has been performed by TALOS+ (37). The protein solutions containing 0.1 M 2-(N-morpholino)ethanesulfonic acid (Mes) secondary structure propensity was determined from the chemical shifts fol- (pH 6.5), 0.2 M ammonium sulfate, and 30% (vol/vol) PEG MME 5000. The data lowing a previously described approach (38), with random-coil reference collection was carried out at PROXIMA1 beamline (SOLEIL, Paris). The dataset chemical shift values corrected for primary sequence, temperature, and was collected at 100 K, using a wavelength of 1.02 Å, and the crystals used for pH effects. data collection were cryo-cooled using 25% (vol/vol) ethylene glycol in the Titrations of 15N-labeled FMN-Ndor1 (oxidized or in the FMNH state) with mother liquor. Data analysis is reported in SI Materials and Methods. 2 unlabeled apo- or oxidized [2Fe-2S]-anamorsin, the N-terminal domain, and oxidized [2Fe-2S]-CIAPIN1-single were performed to follow chemical shift NMR. Standard 1H-detected triple-resonance NMR experiments for backbone changes in 1H-15N HSQC maps. Reversed titrations were performed between resonance assignment were recorded on 0.5- to 1-mM 13C, 15N-labeled samples 15N-labeled or 13C,15N-labeled [2Fe-2S]-anamorsin or [2Fe-2S]-CIAPIN1-single at 298 K ([2Fe-2S]-anamorsin and [2Fe-2S]-CIAPIN1-single) and at 308 K (FMN- 13 and unlabeled FMN-Ndor1, both proteins in the oxidized state, and chemical Ndor1, 1–174 aa), using a Bruker AVANCE 500 MHz spectrometer. C-detected 1 15 13 protonless NMR experiments (26) [CBCACO-in phase antiphase (IPAP), CACO- shift changes followed by H- NHSQCand C-direct detection experiments IPAP, CON-IPAP, CBCACON-IPAP, and CBCANCO-IPAP] acquired on a Bruker (CON, CACO, and CBCACO) after addition of increasing amounts of the AVANCE 700 spectrometer, equipped with a cryogenically cooled probehead unlabeled partner. optimized for 13C-direct detection, were also used for sequence-specificreso- nance assignment (N, C′,Cα,andCβ) of [2Fe-2S]-anamorsin and [2Fe-2S]-CIA- ACKNOWLEDGMENTS. We gratefully acknowledge the Programmi di Ricerca PIN1-single. 13C-direct–detected NMR experiments (26) acquired on 13C,15N- di Rilevante Interesse Nazionale (PRIN) (2009FAKHZT_001), BIO-NMR (Contract ’ ’ labeled [2Fe-2S]-anamorsin allowed us to obtain the backbone resonance as- 261863), WeNMR (Contract 261572), Ministero dell Istruzione, dell Università e CHEMISTRY della Ricerca-Fondo per gli Investimenti della Ricerca di Base (MIUR-FIRB) PRO- signment of all linker residues, with the exception of three (173–175). To TEOMICA (RBRN07BMCT) and Ente Cassa di Risparmio for financial support. identify backbone amide resonances affected by paramagnetic relaxation This work was also supported by Instruct [part of the European Strategy 1 15 1 effects, H- N HSQC, edited by a H inversion recovery and observed in the Forum on Research Infrastructures (ESFRI)] and by national member sub- antiphase component (IR-HSQC-AP), were collected, at 500 MHz, using a 512 × scriptions. Specifically, we thank the European Union ESFRI Instruct Core 100 data point matrix, collected over a 25 × 40 ppm spectral region. A total of Centre Centro di Risonanze Magnetiche (Italy).

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