FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Detailed methods are provided in the online version of this paper and include the following: LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ning Zheng ( nzheng@uw.edu ). Unique and stable reagents generated in this study are available upon request. EXPERIMENTAL MODEL AND SUBJECT DETAILS For DNA extraction, E. coli DH5α was used. For bacmid production, E. coli DH10Bac was used. For baculovirus production and amplification, Sf9 insect cells were used. For protein expression, both E. coli BL21(DE3) and HighFive insect cells were used. For immunoprecipitation in mammalian cells, HEK293T and HEK293 cells were used.

METHOD DETAILS Protein expression and purification

The human IRP2 protein was expressed as a glutathione S-transferase (GST) N-terminal fusion protein in HighFive monolayer insect cells and isolated by glutathione affinity and subsequent anion exchange chromatography after off-column cleavage by tobacco etch virus (TEV) protease. The human FBXL5 and SKP1 proteins were co-expressed and produced in BL21 (DE3) E. coli cells in media supplemented with cysteine and Ferric ammonium citrate (FAC) at the concentrations of 121 mg/L and 25 mg/L, respectively. The plasmid encoding operon suf was co-transformed when needed. The FBXL5-SKP1 complex was purified by nickel affinity and subsequent anion exchange chromatography after off-column cleavage by TEV protease. Human CUL1NTD was expressed as a glutathione S-transferase (GST) N-terminal fusion protein in BL21 (DE3) E. coli cells and purified similarly to IRP2. To assemble the complex of IRP2-FBXL5-SKP1-CUL1NTD for cryo-EM study, the individually isolated proteins were mixed in stoichiometric amounts and subsequently applied to the Superdex-200 gel filtration column (GE Healthcare) in a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 5 mM DTT (dithiothreitol). The mono-dispersive peak in the elution profile contained the tetrameric complex with a mass of ~200 kDa. All the purification procedures were performed at 4°C. The mutants of FBXL5 and IRP2 were expressed and isolated in the same way as the WT proteins. The affinity tag may be left on the proteins for the purposes of different assays.

Cryo-EM sample preparation and data collection

To prepare grids for cryo-EM data collection, an UltraAuFoil R1.2/1.3 grid (Quantifoil Micro Tools GmbH) was glow discharged for 2 minutes at 20 mA with a glow discharge cleaning system (PELCO easiGlow). 3.0 μL of the purified IRP2-FBXL5-SKP1-CUL1NTD complex at 0.5 mg/mL was applied to a freshly glow-discharged grid. After incubating in the chamber at 4 °C and 100% relative humidity, grids were blotted for 4 s with a blotting force of zero, then immediately plunge-frozen in liquid ethane using a Vitrobot Mark IV system (Thermo Fisher Scientific). Data collection was carried out on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV. Automation scheme was implemented using the Leginon software ( Suloway et al., 2005 ) at a nominal magnification of 130 K, resulting a physical pixel size of 1.056 Å. Zero-loss-energy images were acquired on a Gatan K2 Summit direct detector operated in super-resolution counting mode (pixel size in super-resolution mode is 0.528 Å), with the slit width of post-column Gatan Quantum GIF energy filter set to be 20 eV. The dose rate was adjusted to 8.2 electrons per Å 2 per second, and a total dose of 73.8 electrons per Å 2 for each image were fractionated into 60 frames. Data were collected in four sessions with a defocus range of 1.5–3 μm. In total, 5,768 movies were collected with CompuStage in the microscope non-tilted, and a set of 1172 movies were acquired when the CompuStage was tilted 40 degrees.

Image processing and 3D reconstruction

Alignment of movie frames was performed using MotionCor2 ( Zheng et al., 2017 ) through the RELION3.0 pipeline ( Kimanius et al., 2016 ) with images binned 2 in both dimensions by Fourier cropping, resulting in a pixel size of 1.056 Å of the summed images. Dose-weighted summed images were imported into cisTEM ( Grant et al., 2018 ) for manual inspection to remove bad images. The final set of good images contained 5,309 non-tilted and 475 40-degree-tilted images. Around 114,000 particles were selected automatically on a subset of both non-tilted and tilted images after estimation of contrast transfer function using CTFFIND4 ( Rohou and Grigorieff, 2015 ) within cisTEM ( Grant et al., 2018 ) and were subjected to 2D classification to generate 2D averages, which were used later as templates for automatic particle picking. Later steps of data processing were all implemented within the RELION3.0 pipeline ( Kimanius et al., 2016 ). The parameters of contrast transfer function were estimated using cleaned non-dose-weighted motion-corrected sums by GCTF ( Zhang, 2016 ). Template-based automatic particle picking resulted in a set of 2,832,595 particles. GCTF ( Zhang, 2016 ) was used again to perform per-particle estimation of the parameters of contrast transfer function on the automatically picked particles. Particles were then extracted from dose-weighted motion-corrected sums and 3X binned to a box size of 108 pixels. After 3 rounds of reference-free 2D classification, a total of 1,716,405 particles were selected from the original pool. A 3D density map was obtained by employing the 3D initial model job-type within RELION3.0 from a subset of 2D-cleaned-up particles, which was low-pass-filtered to 60 Å and was used as the initial model for 3D classification. A pool of 955,060 particles belonging to the best class from 3D classification were selected, extracted without binning, and subjected to 3D auto-refine without applying a mask, which gave rise to a reconstruction of 3.9 Å. The post-processing procedure with a soft mask generated in RELION3.0 reported an estimated resolution of 3.2 Å. To improve the resolution, CTF refinement ( Zivanov et al., 2018 ) was executed to refine the defocus values for each particle and to calculate the beam-tilt parameters for each separate data-collection session. The resulting particles were polished through the Bayesian polishing approach in RELION3.0 ( Zivanov et al., 2019 ). These polished shiny particles went through another round of focused 3D refinement by imposing a soft mask surrounding the region of interest, which comprises most of mass of the complex, including SKP1, FBXL5, and domains I, II and IV of IRP2. The final reconstruction was measured to be at a resolution of 3.0 Å, and the map was post-processed in RELION3.0, with correction for the modulation transfer function and sharpened by applying a global B-factor of −78 Å 2 that was estimated in the post-process protocol. Reported resolutions are based on the gold-standard FSC (Fourier shell correlation) using the 0.143 criterion ( Rosenthal and Henderson, 2003 ). ResMap ( Kucukelbir et al., 2014 ) was used to determine local resolution.

Model building and refinement

Initial model building was performed on the basis of the resulting map at 3.0 Å after local refinement using a mask to exclude the less-defined flexible regions. Considering that the crystal structures of IRP1 were determined in two different conformations (as an aconitase with PDB ID: 2B3X and as an IRE-binding protein with PDB ID: 3SNP, 3SN2), we used the structures of individual domains in IRP1 as the templates to build the model of IRP2. Models of four domains of IRP1 were fitted into the map as rigid bodies in Chimera ( Pettersen et al., 2004 ) with domain I, II, IV well docked and domain III unassigned due to the lack of density. All the amino acids were changed to the sequence of IRP2 and the model was manually rebuilt in COOT ( Emsley et al., 2010 ). The model of FBXL5 was built de novo in COOT based on the map showing clear side chain densities of the residues in majority, while the crystal structure of SKP1 in GGTase3-FBXL2-SKP1 complex (PDB ID: 6O60) was used as the template for the model building of SKP1. Iterative rounds of real-space refinement in PHENIX ( Adams et al., 2010 ) and manual adjustment in COOT were carried out for model improvement. The final model was evaluated using MolProbity and the cryo-EM data collection, refinement and validation statistics are summarized in Table 1 . Affinity pull-down assay The GST pull-down assay was performed using ~500 μg of purified GST or GST-tagged IRP2 WT or mutant proteins as the bait and ~400 μg of His-tagged FBXL5 WT and mutant proteins. Reaction mixtures were incubated with 100 μL GST beads (GE Healthcare) at 4°C for 1 hour in the binding buffer with 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 5 mM DTT. After extensive wash with binding buffer, the protein complexes on the beads were eluted by 5 mM glutathione. The eluted samples were resolved by SDS-PAGE and analyzed by Coomassie staining. For the pull-down assays with reduced FBXL5, the purified His-tagged FBXL5 WT proteins were treated with 2 mM, 4 mM and 8 mM DT (sodium dithionite) in ambient air or 2 mM DT in an anaerobic glove box (McCoy), and immediately applied to the binding reaction following the same protocol described above. The IRP2-binding-deficient FBXL5 FT (flow through) sample was re-oxidized overnight in ambient air and reapplied to the binding reaction next day ( Figure 5C ). Inputs represent 3–5% of the total amount of proteins used for each reaction. UV/vis absorption spectrometry UV/vis absorption spectra were recorded with 1 μL of protein samples under aerobic conditions at RT on a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific) in a range from 220 to 750 nm. The protein samples were at the concentrations of 8.0 mg/mL or 15.0 mg/mL in the buffer containing 20 mM Tris pH 8.0, 150 mM NaCl and 5 mM DTT. The reduced sample of FBXL5 was prepared anaerobically prior to the aerobic UV/vis spectrum and subsequently re-oxidized overnight in the ambient air. The UV/vis spectrum of the re-oxidized sample was recorded next day. EPR The EPR samples were prepared from 300 μM FBXL5C492Δ-SKP1 protein complex in 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM DTT and 20% (v/v) Glycerol. The reduced EPR sample was prepared in an anaerobic box (McCoy) by the addition of 1.5 mM freshly prepared DT (sodium dithionite). The oxidized sample was prepared aerobically by the addition of 1 mM K 3 Fe(CN) 6 . About 100 μL of solutions were transferred to 4 mm O.D. quartz tubes (Wilmad) and flash-frozen in liquid nitrogen. Atmosphere was removed by freeze-pump-thawing and the samples were flame sealed. X-band EPR spectra were collected on a Bruker EMX spectrometer equipped with an SHQE resonator. The sample temperature was set to 20 K utilizing an Oxford ESR900 liquid-helium flow cryostat. The modulation amplitude was 15 G, microwave power was 32 mW, and the sweep rate was 4 mT s −1 . The magnetic-field axis was calibrated with a teslameter, and the frequency was measured using a frequency counter. The spectrum was simulated using EasySpin ( Stoll and Schweiger, 2006 ).

Native mass spectrometry

FBXL5C492Δ-SKP1 protein complex was buffer exchanged into aqueous 200 mM ammonium acetate at pH 7.0 using centrifugal concentrators (10 kDa MWCO, Spin-X UF, Corning, Inc.) and a centrifuge operated at 4°C. The protein sample used in positive ion mode was also heated using a heating block at ~52°C for one minute prior to analysis. The protein sample was loaded into glass capillaries with inner diameters of 0.78 mm that were pulled to approximately 1 to 3 μm on one end using a micropipette puller (P-97, Sutter Instruments, Novato, CA) for nano electrospray ionization. 10 μM of sample was loaded onto the glass capillary. Electrical contact with the solution was attained by inserting a platinum wire electrode into the wide end of the capillary ( Davidson et al., 2017 ). To minimize impurity carryover between experiments, the electrode was washed with aqueous 25% HCl (v:v) and rinsed with ultrapure (18.2 MΩ) water between samples. Data were acquired using a Waters Synapt G2 HDMS hybrid mass spectrometer ( Giles et al., 2011 ) (Waters Co., Wilmslow, UK) in which the traveling-wave ion mobility cell was replaced with a radio-frequency (RF) confining drift cell ( Allen et al., 2016 ) that contained approximately 1.7 Torr He. The following MS parameters were used for positive ion mode spectrum: capillary voltage, less than 1.0 kV; sampling cone, 70 V; extraction cone, 5 V; source temperature, ~30°C; trap collision energy, 50 V. The following MS parameters were used for negative ion mode spectrum: source temperature, ~30°C; extraction cone, 2 V; trap collision energy, 30 V; sampling cone: 50 V; trap gas flow: 1 ml/min. Mass spectra were analyzed using MassLynx v4.1 (Waters, Co., Milford, MA).

Mammalian expression plasmids

Genestrings of FBXL5C492 WT and 4C>A mutant carrying HA and FLAG tag, BamHI and XhoI sites were custom synthesized and cloned initially into Zero Blunt™ TOPO™ PCR Cloning Kit (Catalog number: 451245). The insert was then restricted from TOPO vector using BamHI and XhoI restriction enzymes and subcloned into in pcDNA5/FRT/TO (Addgene) vector. FLAG tagged IRP2 cloned in pcDNA5/FRT/TO (Addgene) as described in previous study ( Vashisht et al., 2009 ) was used for the indicated experiments.

Mammalian cell culture

Cell lines were purchased from ATCC and routinely monitored for Mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC 30–1012K). HEK293T/HEK293 (ATCC CRL-3216/ATCC CRL-1573) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Corning Life Sciences) and 1% penicillin/streptomycin/L-glutamine (Corning Life Sciences). Cell lines were maintained at 37°C and 5% CO2 in a humidified atmosphere.

Immunoprecipitation and immunoblotting

HEK293T/HEK293 cells were transiently transfected with the indicated plasmids using Polyethylenimine (Polysciences) or Lipofectamine 2000 (Invitrogen). Where indicated, 24 hours after transfection, cells were treated with proteasome inhibitor MG132 (10 μM), or neddylation inhibitor MLN4924 (2.5 μM) for 4 hours before collection. The treatment of MG132 was extended to overnight in antibiotics free media when needed. Cells were harvested and lysed in lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1mM EGTA, 5mM MgCl2, and 0.2% NP-40), supplemented with protease inhibitors (Complete ULTRA, Roche), phosphatase inhibitors (PhosSTOP, Roche), and 1 mM 1,4-Dithiothreitol (DTT) (Sigma-Aldrich). The insoluble fraction was removed by centrifugation (20,000×g) for 15 min at 4°C. Immunoprecipitations of FLAG-tagged proteins were carried out using FLAG-M2 agarose beads (Sigma-Aldrich) for 2 hours at 4°C. The beads were then washed four times in lysis buffer. Immunoprecipitates were eluted in NuPAGE® LDS sample buffer (Thermo Fisher Scientific) supplemented with β-mercaptoethanol (Sigma-Aldrich) and incubated at 95°C for 5 minutes. Whole cell lysates and immunoprecipitates were separated by SDS-PAGE and transferred to either 0.45 μm Immobilon-P PVDF membranes (Millipore Sigma) or nitrocellulose membrane for Western blotting. Transfer efficiency was checked by Ponceau S (Sigma-Aldrich) staining. Membranes were then blocked in 5% milk/TBST for 1 hour at room temperature and incubated with the indicated primary antibodies at 4°C overnight. The detection of proteins was accomplished using the appropriate secondary antibodies conjugated to horseradish peroxidase or for fluorescence (GE Healthcare/Invitrogen) in 5% milk/TBST. Western blots were developed using SuperSignal enhanced chemiluminescence (Thermo Fisher Scientific) using the ImageQuant LAS 4000 (GE Healthcare) image analyzer or LICOR Odyssey imager (LI-COR, Lincoln, NE, USA).

AlphaScreen luminescence proximity assay

AlphaScreen assays for determining and measuring protein-protein and protein-RNA interactions were performed using EnSpire reader (PerkinElmer). Biotinylated Ferritin H IRE (pre-treated as described below) was immobilized to streptavidin coated AlphaScreen donor beads. GST-tagged IRP2 was attached to anti-GST AlphaScreen acceptor beads. The donor and acceptor beads were brought into proximity by the interaction between IRE and IRP2. When excited by a laser beam of 680 nm, the donor beads emit singlet oxygen that activates thioxene derivatives in the acceptor beads, which then release photons of 520–620 nm as the binding signal. Competition assays were performed by titrating the concentrations of the tag-free IRP2 and FBXL5 as competitors in the pre-mixed IRE-IRP2 complex and measuring the half maximal inhibitory concentration (IC 50 ) for competitors based on the dose response curves showing signal recession. The experiments were conducted in triplicates with 0.15 nM biotinylated Ferritin H IRE RNA and 0.15 nM GST-IRP2 in the presence of 5 μg/ml donor and acceptor beads in a buffer of 25mM Hepes pH 7.7, 150 mM NaCl, 5 mM DTT and 0.1% BSA. The concentrations of IRP2 and FBXL5 as competitors ranged from 0.02 nM to 150 nM. IC 50 values were determined using non-linear curve fitting of the dose response curves generated with Prism 8 (GraphPad). The biotinylated Ferritin H IRE RNA was synthesized from Integrated DNA Technologies (IDT). The lyophilized RNA was dissolved in RNase-free water and diluted to 200 μM in concentration. This stock solution was heated at 95°C for 5 min and then cooled in an ice bath for 10 min before being aliquoted. All the aliquots were stored at −80°C to avoid degradation and directly applied to assays after thawing.

QUANTIFICATION AND STATISTICAL ANALYSIS

Protein quantification was done using the Bradford Protein Assay protocol and Bio-rad Protein Assay Dye on a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific) at room temperature.

DATA AND CODE AVAILABILITY

Cryo-EM density map of the IRP2-FBXL5-SKP1 complex has been deposited in the Electron Microscopy Data Bank (EMDB) under the accession code: EMD-21149. Atomic coordinates have been deposited to the Protein Data Bank (PDB) with the accession number PDB: 6VCD.

LEAD CONTACT AND MATERIALS AVAILABILITY

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ning Zheng ( nzheng@uw.edu ). Unique and stable reagents generated in this study are available upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS For DNA extraction, E. coli DH5α was used. For bacmid production, E. coli DH10Bac was used. For baculovirus production and amplification, Sf9 insect cells were used. For protein expression, both E. coli BL21(DE3) and HighFive insect cells were used. For immunoprecipitation in mammalian cells, HEK293T and HEK293 cells were used.

METHOD DETAILS Protein expression and purification

The human IRP2 protein was expressed as a glutathione S-transferase (GST) N-terminal fusion protein in HighFive monolayer insect cells and isolated by glutathione affinity and subsequent anion exchange chromatography after off-column cleavage by tobacco etch virus (TEV) protease. The human FBXL5 and SKP1 proteins were co-expressed and produced in BL21 (DE3) E. coli cells in media supplemented with cysteine and Ferric ammonium citrate (FAC) at the concentrations of 121 mg/L and 25 mg/L, respectively. The plasmid encoding operon suf was co-transformed when needed. The FBXL5-SKP1 complex was purified by nickel affinity and subsequent anion exchange chromatography after off-column cleavage by TEV protease. Human CUL1NTD was expressed as a glutathione S-transferase (GST) N-terminal fusion protein in BL21 (DE3) E. coli cells and purified similarly to IRP2. To assemble the complex of IRP2-FBXL5-SKP1-CUL1NTD for cryo-EM study, the individually isolated proteins were mixed in stoichiometric amounts and subsequently applied to the Superdex-200 gel filtration column (GE Healthcare) in a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 5 mM DTT (dithiothreitol). The mono-dispersive peak in the elution profile contained the tetrameric complex with a mass of ~200 kDa. All the purification procedures were performed at 4°C. The mutants of FBXL5 and IRP2 were expressed and isolated in the same way as the WT proteins. The affinity tag may be left on the proteins for the purposes of different assays.

Cryo-EM sample preparation and data collection

To prepare grids for cryo-EM data collection, an UltraAuFoil R1.2/1.3 grid (Quantifoil Micro Tools GmbH) was glow discharged for 2 minutes at 20 mA with a glow discharge cleaning system (PELCO easiGlow). 3.0 μL of the purified IRP2-FBXL5-SKP1-CUL1NTD complex at 0.5 mg/mL was applied to a freshly glow-discharged grid. After incubating in the chamber at 4 °C and 100% relative humidity, grids were blotted for 4 s with a blotting force of zero, then immediately plunge-frozen in liquid ethane using a Vitrobot Mark IV system (Thermo Fisher Scientific). Data collection was carried out on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operated at 300 kV. Automation scheme was implemented using the Leginon software ( Suloway et al., 2005 ) at a nominal magnification of 130 K, resulting a physical pixel size of 1.056 Å. Zero-loss-energy images were acquired on a Gatan K2 Summit direct detector operated in super-resolution counting mode (pixel size in super-resolution mode is 0.528 Å), with the slit width of post-column Gatan Quantum GIF energy filter set to be 20 eV. The dose rate was adjusted to 8.2 electrons per Å 2 per second, and a total dose of 73.8 electrons per Å 2 for each image were fractionated into 60 frames. Data were collected in four sessions with a defocus range of 1.5–3 μm. In total, 5,768 movies were collected with CompuStage in the microscope non-tilted, and a set of 1172 movies were acquired when the CompuStage was tilted 40 degrees.

Image processing and 3D reconstruction

Alignment of movie frames was performed using MotionCor2 ( Zheng et al., 2017 ) through the RELION3.0 pipeline ( Kimanius et al., 2016 ) with images binned 2 in both dimensions by Fourier cropping, resulting in a pixel size of 1.056 Å of the summed images. Dose-weighted summed images were imported into cisTEM ( Grant et al., 2018 ) for manual inspection to remove bad images. The final set of good images contained 5,309 non-tilted and 475 40-degree-tilted images. Around 114,000 particles were selected automatically on a subset of both non-tilted and tilted images after estimation of contrast transfer function using CTFFIND4 ( Rohou and Grigorieff, 2015 ) within cisTEM ( Grant et al., 2018 ) and were subjected to 2D classification to generate 2D averages, which were used later as templates for automatic particle picking. Later steps of data processing were all implemented within the RELION3.0 pipeline ( Kimanius et al., 2016 ). The parameters of contrast transfer function were estimated using cleaned non-dose-weighted motion-corrected sums by GCTF ( Zhang, 2016 ). Template-based automatic particle picking resulted in a set of 2,832,595 particles. GCTF ( Zhang, 2016 ) was used again to perform per-particle estimation of the parameters of contrast transfer function on the automatically picked particles. Particles were then extracted from dose-weighted motion-corrected sums and 3X binned to a box size of 108 pixels. After 3 rounds of reference-free 2D classification, a total of 1,716,405 particles were selected from the original pool. A 3D density map was obtained by employing the 3D initial model job-type within RELION3.0 from a subset of 2D-cleaned-up particles, which was low-pass-filtered to 60 Å and was used as the initial model for 3D classification. A pool of 955,060 particles belonging to the best class from 3D classification were selected, extracted without binning, and subjected to 3D auto-refine without applying a mask, which gave rise to a reconstruction of 3.9 Å. The post-processing procedure with a soft mask generated in RELION3.0 reported an estimated resolution of 3.2 Å. To improve the resolution, CTF refinement ( Zivanov et al., 2018 ) was executed to refine the defocus values for each particle and to calculate the beam-tilt parameters for each separate data-collection session. The resulting particles were polished through the Bayesian polishing approach in RELION3.0 ( Zivanov et al., 2019 ). These polished shiny particles went through another round of focused 3D refinement by imposing a soft mask surrounding the region of interest, which comprises most of mass of the complex, including SKP1, FBXL5, and domains I, II and IV of IRP2. The final reconstruction was measured to be at a resolution of 3.0 Å, and the map was post-processed in RELION3.0, with correction for the modulation transfer function and sharpened by applying a global B-factor of −78 Å 2 that was estimated in the post-process protocol. Reported resolutions are based on the gold-standard FSC (Fourier shell correlation) using the 0.143 criterion ( Rosenthal and Henderson, 2003 ). ResMap ( Kucukelbir et al., 2014 ) was used to determine local resolution.

Model building and refinement

Initial model building was performed on the basis of the resulting map at 3.0 Å after local refinement using a mask to exclude the less-defined flexible regions. Considering that the crystal structures of IRP1 were determined in two different conformations (as an aconitase with PDB ID: 2B3X and as an IRE-binding protein with PDB ID: 3SNP, 3SN2), we used the structures of individual domains in IRP1 as the templates to build the model of IRP2. Models of four domains of IRP1 were fitted into the map as rigid bodies in Chimera ( Pettersen et al., 2004 ) with domain I, II, IV well docked and domain III unassigned due to the lack of density. All the amino acids were changed to the sequence of IRP2 and the model was manually rebuilt in COOT ( Emsley et al., 2010 ). The model of FBXL5 was built de novo in COOT based on the map showing clear side chain densities of the residues in majority, while the crystal structure of SKP1 in GGTase3-FBXL2-SKP1 complex (PDB ID: 6O60) was used as the template for the model building of SKP1. Iterative rounds of real-space refinement in PHENIX ( Adams et al., 2010 ) and manual adjustment in COOT were carried out for model improvement. The final model was evaluated using MolProbity and the cryo-EM data collection, refinement and validation statistics are summarized in Table 1 . Affinity pull-down assay The GST pull-down assay was performed using ~500 μg of purified GST or GST-tagged IRP2 WT or mutant proteins as the bait and ~400 μg of His-tagged FBXL5 WT and mutant proteins. Reaction mixtures were incubated with 100 μL GST beads (GE Healthcare) at 4°C for 1 hour in the binding buffer with 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 5 mM DTT. After extensive wash with binding buffer, the protein complexes on the beads were eluted by 5 mM glutathione. The eluted samples were resolved by SDS-PAGE and analyzed by Coomassie staining. For the pull-down assays with reduced FBXL5, the purified His-tagged FBXL5 WT proteins were treated with 2 mM, 4 mM and 8 mM DT (sodium dithionite) in ambient air or 2 mM DT in an anaerobic glove box (McCoy), and immediately applied to the binding reaction following the same protocol described above. The IRP2-binding-deficient FBXL5 FT (flow through) sample was re-oxidized overnight in ambient air and reapplied to the binding reaction next day ( Figure 5C ). Inputs represent 3–5% of the total amount of proteins used for each reaction. UV/vis absorption spectrometry UV/vis absorption spectra were recorded with 1 μL of protein samples under aerobic conditions at RT on a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific) in a range from 220 to 750 nm. The protein samples were at the concentrations of 8.0 mg/mL or 15.0 mg/mL in the buffer containing 20 mM Tris pH 8.0, 150 mM NaCl and 5 mM DTT. The reduced sample of FBXL5 was prepared anaerobically prior to the aerobic UV/vis spectrum and subsequently re-oxidized overnight in the ambient air. The UV/vis spectrum of the re-oxidized sample was recorded next day. EPR The EPR samples were prepared from 300 μM FBXL5C492Δ-SKP1 protein complex in 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM DTT and 20% (v/v) Glycerol. The reduced EPR sample was prepared in an anaerobic box (McCoy) by the addition of 1.5 mM freshly prepared DT (sodium dithionite). The oxidized sample was prepared aerobically by the addition of 1 mM K 3 Fe(CN) 6 . About 100 μL of solutions were transferred to 4 mm O.D. quartz tubes (Wilmad) and flash-frozen in liquid nitrogen. Atmosphere was removed by freeze-pump-thawing and the samples were flame sealed. X-band EPR spectra were collected on a Bruker EMX spectrometer equipped with an SHQE resonator. The sample temperature was set to 20 K utilizing an Oxford ESR900 liquid-helium flow cryostat. The modulation amplitude was 15 G, microwave power was 32 mW, and the sweep rate was 4 mT s −1 . The magnetic-field axis was calibrated with a teslameter, and the frequency was measured using a frequency counter. The spectrum was simulated using EasySpin ( Stoll and Schweiger, 2006 ).

Native mass spectrometry

FBXL5C492Δ-SKP1 protein complex was buffer exchanged into aqueous 200 mM ammonium acetate at pH 7.0 using centrifugal concentrators (10 kDa MWCO, Spin-X UF, Corning, Inc.) and a centrifuge operated at 4°C. The protein sample used in positive ion mode was also heated using a heating block at ~52°C for one minute prior to analysis. The protein sample was loaded into glass capillaries with inner diameters of 0.78 mm that were pulled to approximately 1 to 3 μm on one end using a micropipette puller (P-97, Sutter Instruments, Novato, CA) for nano electrospray ionization. 10 μM of sample was loaded onto the glass capillary. Electrical contact with the solution was attained by inserting a platinum wire electrode into the wide end of the capillary ( Davidson et al., 2017 ). To minimize impurity carryover between experiments, the electrode was washed with aqueous 25% HCl (v:v) and rinsed with ultrapure (18.2 MΩ) water between samples. Data were acquired using a Waters Synapt G2 HDMS hybrid mass spectrometer ( Giles et al., 2011 ) (Waters Co., Wilmslow, UK) in which the traveling-wave ion mobility cell was replaced with a radio-frequency (RF) confining drift cell ( Allen et al., 2016 ) that contained approximately 1.7 Torr He. The following MS parameters were used for positive ion mode spectrum: capillary voltage, less than 1.0 kV; sampling cone, 70 V; extraction cone, 5 V; source temperature, ~30°C; trap collision energy, 50 V. The following MS parameters were used for negative ion mode spectrum: source temperature, ~30°C; extraction cone, 2 V; trap collision energy, 30 V; sampling cone: 50 V; trap gas flow: 1 ml/min. Mass spectra were analyzed using MassLynx v4.1 (Waters, Co., Milford, MA).

Mammalian expression plasmids

Genestrings of FBXL5C492 WT and 4C>A mutant carrying HA and FLAG tag, BamHI and XhoI sites were custom synthesized and cloned initially into Zero Blunt™ TOPO™ PCR Cloning Kit (Catalog number: 451245). The insert was then restricted from TOPO vector using BamHI and XhoI restriction enzymes and subcloned into in pcDNA5/FRT/TO (Addgene) vector. FLAG tagged IRP2 cloned in pcDNA5/FRT/TO (Addgene) as described in previous study ( Vashisht et al., 2009 ) was used for the indicated experiments.

Mammalian cell culture

Cell lines were purchased from ATCC and routinely monitored for Mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC 30–1012K). HEK293T/HEK293 (ATCC CRL-3216/ATCC CRL-1573) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Corning Life Sciences) and 1% penicillin/streptomycin/L-glutamine (Corning Life Sciences). Cell lines were maintained at 37°C and 5% CO2 in a humidified atmosphere.

Immunoprecipitation and immunoblotting

HEK293T/HEK293 cells were transiently transfected with the indicated plasmids using Polyethylenimine (Polysciences) or Lipofectamine 2000 (Invitrogen). Where indicated, 24 hours after transfection, cells were treated with proteasome inhibitor MG132 (10 μM), or neddylation inhibitor MLN4924 (2.5 μM) for 4 hours before collection. The treatment of MG132 was extended to overnight in antibiotics free media when needed. Cells were harvested and lysed in lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1mM EGTA, 5mM MgCl2, and 0.2% NP-40), supplemented with protease inhibitors (Complete ULTRA, Roche), phosphatase inhibitors (PhosSTOP, Roche), and 1 mM 1,4-Dithiothreitol (DTT) (Sigma-Aldrich). The insoluble fraction was removed by centrifugation (20,000×g) for 15 min at 4°C. Immunoprecipitations of FLAG-tagged proteins were carried out using FLAG-M2 agarose beads (Sigma-Aldrich) for 2 hours at 4°C. The beads were then washed four times in lysis buffer. Immunoprecipitates were eluted in NuPAGE® LDS sample buffer (Thermo Fisher Scientific) supplemented with β-mercaptoethanol (Sigma-Aldrich) and incubated at 95°C for 5 minutes. Whole cell lysates and immunoprecipitates were separated by SDS-PAGE and transferred to either 0.45 μm Immobilon-P PVDF membranes (Millipore Sigma) or nitrocellulose membrane for Western blotting. Transfer efficiency was checked by Ponceau S (Sigma-Aldrich) staining. Membranes were then blocked in 5% milk/TBST for 1 hour at room temperature and incubated with the indicated primary antibodies at 4°C overnight. The detection of proteins was accomplished using the appropriate secondary antibodies conjugated to horseradish peroxidase or for fluorescence (GE Healthcare/Invitrogen) in 5% milk/TBST. Western blots were developed using SuperSignal enhanced chemiluminescence (Thermo Fisher Scientific) using the ImageQuant LAS 4000 (GE Healthcare) image analyzer or LICOR Odyssey imager (LI-COR, Lincoln, NE, USA).

AlphaScreen luminescence proximity assay

AlphaScreen assays for determining and measuring protein-protein and protein-RNA interactions were performed using EnSpire reader (PerkinElmer). Biotinylated Ferritin H IRE (pre-treated as described below) was immobilized to streptavidin coated AlphaScreen donor beads. GST-tagged IRP2 was attached to anti-GST AlphaScreen acceptor beads. The donor and acceptor beads were brought into proximity by the interaction between IRE and IRP2. When excited by a laser beam of 680 nm, the donor beads emit singlet oxygen that activates thioxene derivatives in the acceptor beads, which then release photons of 520–620 nm as the binding signal. Competition assays were performed by titrating the concentrations of the tag-free IRP2 and FBXL5 as competitors in the pre-mixed IRE-IRP2 complex and measuring the half maximal inhibitory concentration (IC 50 ) for competitors based on the dose response curves showing signal recession. The experiments were conducted in triplicates with 0.15 nM biotinylated Ferritin H IRE RNA and 0.15 nM GST-IRP2 in the presence of 5 μg/ml donor and acceptor beads in a buffer of 25mM Hepes pH 7.7, 150 mM NaCl, 5 mM DTT and 0.1% BSA. The concentrations of IRP2 and FBXL5 as competitors ranged from 0.02 nM to 150 nM. IC 50 values were determined using non-linear curve fitting of the dose response curves generated with Prism 8 (GraphPad). The biotinylated Ferritin H IRE RNA was synthesized from Integrated DNA Technologies (IDT). The lyophilized RNA was dissolved in RNase-free water and diluted to 200 μM in concentration. This stock solution was heated at 95°C for 5 min and then cooled in an ice bath for 10 min before being aliquoted. All the aliquots were stored at −80°C to avoid degradation and directly applied to assays after thawing.

Supplementary Material 2