Enzyme activity was measured using the E. coli isc proteins IscS, IscU, HscA, HscB, and apo Fdx. This in vitro assay is a sensitive method to follow the enzymatic rates by detecting the absorbance of a reporter acceptor as a function of time (Adinolfi et al., 2009; Prischi et al., 2010b). We monitored the signal at 458 nm because this is the characteristic wavelength for the absorbance of FeS clusters on Fdx. Because we work with a complex multi-component system, we first optimized the relative concentrations of the components. The concentration of apo Fdx, which was used as the final cluster acceptor, was set to 50 μM which is a value that allows easy measurement of the absorbance signal from the cluster. IscS was set to catalytic concentration (1 μM) to minimize contributions to the spectrum of unspecific iron-thiolate polysulfides bound to IscS (Bonomi et al., 1985). The presence of the antioxidant DTT is necessary to provide the reducing equivalents required for cluster generation and to regenerate the prosthetic group pyridoxal phosphate (Urbina et al., 2001). We started with 1 μM IscU, 1 μM HscA, and 1 μM HscB, 250 μM Cys, 150 μM ATP, 25 μM Fe²⁺ and, unless explicitely indicated, 10 mM Mg²⁺. We measured the slope of the curves after the lag phase when present to compare the rates semi-quantitatively.

Increasing IscU concentrations (1, 5, 10, 20 μM) enhanced the reconstitution rate and shortened the lag phase, reaching a plateau above 5–10 μM (Figure 1A). This is reasonable since increase of IscU concentration facilitates formation of the IscS-IscU complex and accelerates the reaction. We thus adopted 8 μM concentrations of IscU in the following experiments to ensure high activity but, at the same time, avoid contributions from holo-IscU to absorbance. Progressive increase of HscB concentration caused a clear reduction of the reaction rates (Figure 1B). A 3 μM HscB concentration was adopted in the following experiments to ensure a small excess of protein with respect to IscS in a range of concentrations where inhibition is not dominant. The ATPase activity of HscA was independently checked by a spectrophotometric method that allows the quantification of inorganic phosphate release (data not shown). Variation of the HscA concentration in the range 1–5 μM led to no change of the cluster formation rates. Above ~5 μM, we observed a deep decrease of the rates (Figure 1C). A 2 μM concentration was thus adopted in the following measurement to ensure a small excess of protein with respect to IscS. Finally, we screened the effect of ATP varying it from 0 to 1 mM in the absence and in the presence of 10 mM Mg²⁺. When no Mg²⁺ was added we observed a dramatic reduction of the rates up to almost complete inhibition of the reaction (Figure 1D). The effect was drastically reduced and practically abolished at 10 mM Mg²⁺ which are the concentrations typically used for this assay because close to the cellular conditions.

These results reproduce a previous study which was however carried out mainly in the absence of IscS (Iametti et al., 2015). A plausible way to explain the effect of ATP, which had been left with no explanation, is that ATP is well known to chelate iron (Mansour et al., 1985; Patchornik et al., 2000). It would then result in depletion of Fe²⁺ from solution. Mg²⁺, the counter ion always used with chaperones, is thus particularly important to compete with Fe²⁺ binding.

To understand the factors which determine inhibition of HscB and HscA, we explored the effects of each component by introducing them individually and in pair in the absence and in the presence of ATP (Figure 2A). We used again Fdx as the reporter, 1 μM of IscS and 8 μM of IscU, in the presence of 250 μM Cys, 25 μM Fe²⁺, and 10 mM Mg²⁺. In the absence of ATP, introduction of HscA (10 μM) does not produce appreciable effects on the enzymatic rates. Addition of HscB (10 μM) remarkably inhibits the rates, while co-addition of HscA and HscB produces a further drop. In the presence of ATP, addition of HscA increases the rates in a comparable way than when adding only ATP. Addition of HscB, alone or together with HscA and ATP appreciably reduces the rates. This tells us that the effect is mainly due to HscB, whether in the presence or absence of HscA.

We thus decided to focus on HscB and leave for future studies the more complex effect of HscA, which is further complicated by ATP. We first repeated the screening of HscB concentrations, but this time in the absence of ATP and HscA. We observed a clear progressive drop of the rates (Figure 2B left panel). Plotting the slope of the curves vs. the concentration we obtained a rate decrease, which reaches a plateau between 20 and 40 μM (Figure 2B right panel). We wondered if the effect could be due to the protein reporter given that Fdx is somewhat a special case because it interacts specifically with IscS (Yan et al., 2013). We thus repeated the measurements using IscU or aconitase (Beinert et al., 1996) as reporter (35 μM). Addition of HscB decreased both rates as in the previous cases (Figures 2C,D). The effect is thus independent from the reporter.

We then measured the desulfurase activity by detecting the progressive disappearance of cysteine and concomitant appearance of alanine by mass spectrometry in the absence of IscU at different time points which allowed us to follow the rates of desulfuration. We used the same concentrations as in the previous assays. Once initiating the reaction by adding the substrate, mixtures of acetic acid/acetonitrile were used to stop it. The presence of HscB slows down the reaction resulting in slower rates (Figure 2E).

Altogether, these results tell us that the inhibitory effect is mainly linked to HscB but it is not only due to the interaction between IscU and HscB as previously suggested (Hoff et al., 2000; Iametti et al., 2015): HscB has also effects on the desulfurase step and thus on IscS.

Although not previously reported, a possible working hypothesis to explain the observed effect is that HscB binds also to IscS and that this interferes with the desulfuration step. To test the hypothesis, we carried out pull-down and size exclusion chromatography assays but both proved inconclusive, as it can be expected for weak complexes. We thus used microscale thermophoresis (MST) which is a sensitive means to measure weak interactions (Jerabek-Willemsen et al., 2014). The thermophoretic movement of the fluorescently labeled IscS was assessed by measuring the fluorescence distribution inside a capillary and the microscopic temperature gradient generated by a laser. The binding curve was obtained by plotting the fluorescence vs. the logarithm of the progressively diluted concentrations of HscB (Figure 3A). The data obtained by different values of light emitting diode (LED) resulted in a dissociation constant (K_D) of 10–30 μM assuming a 1:1 stoichiometry. These affinities are low but comparable to those observed for other components of the isc machine (Prischi et al., 2010b) and, more importantly, to that observed for the IscU/HscB interaction (~10 μM, Hoff et al., 2000). To confirm the interaction with an independent method, we used chemical cross-linking, a technique able to capture weak or transient protein-protein interactions (Watson et al., 2012). We added bis[sulfosuccinimidyl]suberate, a cross-linking agent that reacts with primary amino groups, to a mixture containing HscB and IscS. SDS-PAGE revealed the presence of a new species at around 65 KDa that corresponds to HscB-IscS covalently bound (Figure 3B).

We mapped the residues of HscB involved by ¹⁵N HSQC NMR experiments recorded on a ¹⁵N labeled HscB sample titrated with unlabeled IscS up to a 1:2 HscB:IscS molar ratio. At a 1:1 IscS/HscB molar ratio, many peaks, amongst which those from the J-domain, do not shift (Figure 3C). This suggests that IscS has a minimal effect on these regions of HscB. Other resonances (L22, H63, Q95, R99, E104, E106, E111, I118, K119, M124, L157, A161, and D170) have small but detectable chemical shift changes indicative of environmental changes in the presence of IscS. These residues are mainly located on the C-terminus and map on the surface facing the J-domain. A third set of residues (I105, I118, F125, H129, L131, L136, D139, L151, D155, R158, E162, and K167) broaden significantly. At the end of the titration, the HscB spectrum disappears almost completely as expected for the formation of a complex with a molecular weight of 130 kDa (assuming a 1:1 stoichiometry). Complexes of this size are not usually observable without deuteration because their large correlation times (τ_c) causes excessive line broadening of the resonances. To ensure that these variations involve a direct interaction rather than a conformational change which could propagate far from the surface of contact, we performed cross saturation experiments (Takahashi et al., 2000) using deuterated HscB. The following residues were observed to have an effect: L22, Q49, Q56, A80, Q95, D110, E111, K119, S160, Q163, E166, D170, and F171. Most of these residues are close in space and located in the C-terminus of the approximately L-shaped HscB structure and form an exposed patch (Figure 3D). We validated the surface of interaction on HscB by designing two acid-to-base mutants which can be expected to affect binding (HscB_E111K/E115K and HscB_E165K/E166K). Their circular dichroism spectra are superposable on that of the wild-type proving that they retain the fold (Figure S1). The enzymatic rates in the presence of the individual mutants led to effects that approach the behavior observed in the absence of HscB.

Overall, these results validate the binding interface on HscB. We can thus conclude that there is a weak but specific binding between HscB and IscS.

In turn, we tested the ability of ad hoc designed IscS mutants (IscS_R39E/W45E, IscS_K101E/K105E, IscS_R220E/R223E/R225E, IscS_I314E/M315E, and IscS_E334S/R340S) which affect different regions of the protein to identify the surface of IscS interacting with HscB. Circular dichroism supports that the mutants are all correctly folded as expected from their location in exposed regions of the enzyme (Prischi et al., 2010b). Among them, IscS_R220E/R223E/R225E, IscS_R39E/W45E, and IscS_K101E/K105E leave mostly unaffected the spectrum of ¹⁵N HscB (Figure 4). This means that mutations of these residues results in complete or partial abolishment of binding. Conversely, the spectrum is affected by IscS_I314E/M315E and IscS_E334S/R340S titration comparably to wild-type, indicating that these residues are not involved in the interaction. A similar behavior has been reported for the frataxin ortholog CyaY (Prischi et al., 2010b) and for Fdx (Yan et al., 2013) which also form complexes with IscS with micromolar affinities. These results thus suggest that HscB binds IscS in a site overlapping with that observed for these proteins.

To validate the hypothesis, we titrated a sample containing the HscB/IscS complex with CyaY (up to a 1:1:3 molar ratio) or Fdx (up to a ratio 1:1:3). We observed that introduction of CyaY causes the progressive reappearance of the spectrum of HscB, consistent with almost complete displacement of IscS at 1:1:1 (Figure 5, top and middle panels). Titration of the HscB/IscS complex with Fdx also regenerates the spectrum of HscB but at a higher molar ratio: at a 1:1:3 ratio of IscS/HscB/Fdx, the spectrum of HscB is only partially restored (Figure 5, top and bottom panels). This is in agreement with the relative binding affinities (Prischi et al., 2010b; Yan et al., 2013) and confirms that the interaction between IscS and HscB involves the same site which hosts CyaY and Fdx. The site involves a cleft formed between the two protomers of the IscS dimer and contains PLP and the catalytic center (Cupp-Vickery et al., 2003).

Finally, we checked if the HscB/IscS complex is compatible or mutually exclusive with IscU binding. We added unlabeled HscB to ¹⁵N labeled IscU (1:0.4 IscU:HscB) and observed the typical chemical shifts expected for binding (Figure 6, Left). We then added unlabeled IscS (1:0.4:0.4) and observed further disappearance of the spectrum. Since this could simply mean a displacement of HscB, we also titrated labeled HscB with unlabeled IscU (1:1), to which we added unlabeled IscS (1:1:1). Also in this case the spectrum disappears almost completely (Figure 6, Right). Titration of HscB with IscS first and then with IscU led to the same result (data not shown). These results conclusively indicate that interaction between HscB and IscU is compatible with IscS binding, a hypothesis not previously considered.

Proteins were subcloned by PCR from bacterial genomic DNA and individually expressed from pET-derived plasmid vectors as fusion proteins with His-tagged glutathione-Stransferase (GST) and a cleavage site for Tobacco Etch Virus (TEV) protease. They were expressed and purified from E. coli strain BL21(DE3) as previously described (Adinolfi et al., 2002; Prischi et al., 2010a). Cells were inoculated at 37°C in LB medium with kanamycin (30 mg/l), induced for overnight at 18°C by the addition of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) after the cultures reached an optical density (OD) at 600 nm of 0.6–0.8. Cell pellets were centrifuged, resuspended in a lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM imidazole, Igepal, DNAse I, lysozyme, antiprotease, 1 mM TCEP) and frozen. Cell pellets were then thawed and sonicated and centrifuged. Proteins were purified by affinity chromatography (using Ni-nitrilotriacetic agarose), eluted and cleaved overnight from His, GST-tag by TEV protease dialysing in 20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM DTT. Further purification was carried out by gel filtration chromatography on a 16/60 Superdex G75 column. Samples were eluted in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1 mM TCEP and monitoring absorbance at 280 nm. Protein concentration was determined by UV spectroscopy. Protein purity was checked by SDS-PAGE after each step of the purification. ¹⁵N labeled samples for NMR measurements were grown in minimal medium using (¹⁵NH₄)₂SO₄ as the sole source of nitrogen while ¹⁵N,²H labeled samples were grown in minimal medium containing D₂O and using (¹⁵NH₄)₂SO₄.

Enzymatic cluster formation was achieved under strict anaerobic conditions in a Belle chamber kept under nitrogen atmosphere. The reaction was followed by absorbance spectroscopy using a Cary 50 Bio Spectrophotometer (Varian). Absorbance variations at 458 or 406 nm were measured as a function of time. A solution of 50 μM of apoFdx or 35 μM aconitase was incubated in sealed cuvettes typically using 3 mM DTT, IscU, HscA, HscB, 1 μM IscS, and 25 μM Fe(NH₄)₂(SO₄)₂ for 30 min in 20 mM Tris-HCl pH 8 and 150 mM NaCl. The reaction was initiated by adding 250 μM of the substrate L-cysteine and when specified 150 μM ATP. Each experiment was repeated at least 3 times on different batches of proteins and by two different researchers. To simplify the analysis, we took the initial slopes of the curves (absorbance vs. time) to qualitatively compare the time courses although some curves have a lag phase which likely reflects diffusion of the components.

MST measurements were performed using a NanoTemper Monolith NT.115 instrument. IscS samples were labeled with the amine-reactive dye NT-647 using the Monolith NT.115 Protein Labeling Kit RED-NHS. The reaction mixture was incubated overnight at 4°C. Labeling levels (in the range of 0.67 dye molecules per IscS) were determined using ε₂₈₀(IscS) 41,370 M⁻¹cm⁻¹ and ε₆₅₀(dye) 250,000 M⁻¹cm⁻¹. HscB was dissolved to a final concentration of 600 μM in a buffer containing 20 mM Tris-HCl pH 8, 150 mM NaCl, 0.5 mM TCEP, 0.05% Tween-20 and labeled IscS at a concentration of 52 nM. The stock solution was then serially diluted to 1:1 using the same buffer to give 12 working solutions with different HscB concentration but the same fluorophore concentration. The solutions were then loaded into hydrophilic capillaries and MST measurement were made at 25°C using 40–60% light-emitting diode power and 17% of infrared-laser power. Each measurement was repeated at least 3 times.

Alanine formation was obtained with 1 μM IscS, 3 mM DTT, and 250 μM cysteine in 20 mM Tris-HCl at pH 8 and 150 mM NaCl. When necessary, 3 μM HscB was added. The reaction was stopped with a 150 μl mixture acetic acid (0.1%)/acetonitrile (100%) after 0, 10, 20, 40, and 60 min. Determination of alanine and cysteine was performed by HR-LCMS using a Thermo Accela Pump and Pal Autosampler coupled to a Thermo EXactive. Chromatographic separation was performed on a ODS Hypersil 150 × 2.1 mm 3u column maintained at room temperature. A 10 min gradient was employed using a mobile phase A with 0.1% formic acid in water and a mobile phase B in 0.1% formic acid in acetonitrile.

NMR spectra were acquired on Bruker AVANCE spectrometers operating at 600, 700, and 800 MHz proton frequencies. Typically, measurements were carried out at 298 K in 20 mM Tris-HCl pH 8, 150 mM NaCl, and 2 mM DTT using a 0.1 mM uniformly ¹⁵N-enriched protein. Water suppression was achieved by using WATERGATE and HSQC experiments were recorded. The spectra were processed by using the NMRPipe program and analyzed by Ccpnmr. Cross saturation experiments were carried out on ²H,¹⁵N-labeled HscB sample and using TROSY detection. Spectral assignment of HscB was based on the BMRB deposition.

A mixture of 5 μM IscS and 30 μM HscB was prepared in PBS buffer. Bis[sulfosuccinimidyl]suberate was added to the protein sample to a final concentration of 1.3 mM. The reaction mixture was incubated at room temperature for 30 min and quenched by adding Tris-HCl at pH 8 to a final concentration of 20 mM. The quenching reaction was incubated at room temperature for 15 min. SDS-PAGE was run to observe the cross-linked species.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.