Structural and Molecular Characterization of Iron-sensingHemerythrin-like Domain within F-box and Leucine-richRepeat Protein 5 (FBXL5)□S �

Received for publication, September 28, 2011, and in revised form, November 18, 2011 Published, JBC Papers in Press, January 17, 2012, DOI 10.1074/jbc.M111.308684

Joel W. Thompson‡1, Ameen A. Salahudeen‡, Srinivas Chollangi‡, Julio C. Ruiz‡2, Chad A. Brautigam‡,Thomas M. Makris§, John D. Lipscomb§3, Diana R. Tomchick‡, and Richard K. Bruick‡4

From the ‡Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390 and the§Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

Background: FBXL5 is required for proper regulation of cellular iron homeostasis.Results: FBXL5 contains a structurally characterized hemerythrin-like domain.Conclusion: The atypical features of the hemerythrin-like domain facilitate its role as a metabolic sensor.Significance: FBXL5 is the only identified mammalian protein containing a hemerythrin-like domain. Resolution of the struc-ture of the domain provides mechanistic insights into its iron and oxygen responsiveness.

Mammalian cells maintain iron homeostasis by sensingchanges in bioavailable iron levels and promoting adaptiveresponses. FBXL5 is a subunit of an E3 ubiquitin ligase complexthat mediates the stability of iron regulatory protein 2, animportant posttranscriptional regulator of several genesinvolved in ironmetabolism. The stability of FBXL5 is regulatedin an iron- and oxygen-responsivemanner, contingent upon thepresence of its N-terminal domain. Here we present the atomicstructure of theFBXL5Nterminus, a hemerythrin-like�-helicalbundle fold not previously observed in mammalian proteins.The core of this domain employs an unusual assortment ofamino acids necessary for the assembly and sensing propertiesof its diiron center. These regulatory features govern the acces-sibility of a mapped sequence required for proteasomal degra-dation of FBXL5. Detailed molecular and structural character-ization of the ligand-responsive hemerythrin domain providesinsights into themechanismsbywhichFBXL5 serves as a uniquemammalian metabolic sensor.

Because both iron deficiency and iron overload can be dele-terious, it is imperative that cells maintain proper iron homeo-stasis (1). Cellular iron homeostasis is regulated in part by iron

regulatory protein 2 (IRP2),5 which posttranscriptionally regu-lates a cohort of ironmetabolism genes (2). IRP2 controls eitherthe translation or the stability of its targetmRNAs by binding toRNA hairpin structures, known as iron-responsive elements (3,4). IRP2 accumulates under iron- or oxygen-deficient condi-tions but is polyubiquitinated and degraded by the proteasomewhen iron and oxygen are plentiful (5–7). The physiologicalimportance of IRP2 is reflected in the observed phenotypesarising from targeted gene deletion, including anemia andmildneurodegeneration (8–11). Proper IRP2 regulation necessi-tates that cells sense both iron and oxygen availability and ameans by which such information affects an appropriatechange in IRP2 stability. However, the mechanisms by whichIRP2 is regulated in an iron- and oxygen-dependentmanner areonly beginning to be delineated.Recently, we and others reported that an E3 ubiquitin ligase

complex containing F-box and leucine-rich repeat protein 5(FBXL5) regulates IRP2 stability (12, 13). A reduction in FBXL5expression leads to inappropriate accumulation of IRP2 andsubsequentmisregulation of IRP2 target genes in cultured cells,indicating that FBXL5 contributes to the maintenance of ironhomeostasis. Notably, FBXL5 is regulated in an inverse fashionto IRP2 as it is stabilized under iron-replete conditions andpreferentially degraded when iron or oxygen becomes limiting.Domain mapping studies revealed that the N terminus ofFBXL5 is required for its iron-dependent regulation. Bioinfor-matic analysis predicted that the N-terminal region of FBXL5encodes a hemerythrin-like domain (12, 13).Hemerythrin (Hr) domains are characterized by an �-helical

bundle fold that commonly contains a diiron center (14),although other metal centers are possible (Protein Data Bank(PDB) ID 2P0N) (16–18). These iron atoms are redox-activeand can switch between the fully oxidized diferric (met) state, apartially reduced semi-met state, and a fully reduced diferrous(deoxy) state. In canonical Hr domains, one iron atom (Fe1) is

� This article was selected as a Paper of the Week.□S This article contains supplemental Figs. S1–S7 and Tables S1–S5.The atomic coordinates and structure factors (codes 3V5X, 3V5Y, and 3V5Z)

have been deposited in the Protein Data Bank, Research Collaboratory forStructural Bioinformatics, Rutgers University, New Brunswick, NJ(http://www.rcsb.org/).

1 Supported by the Graduate Programs Initiative from the University of TexasBoard of Regents.

2 A Howard Hughes Medical Institute Scholar. Supported by a Sara and FrankMcKnight Graduate Student Fellowship.

3 Supported by National Institutes of Health Grant GM24689.4 The Michael L. Rosenberg Scholar in Medical Research. Supported by a

Career Award in the Biomedical Sciences from the Burroughs WellcomeFund, the Robert A. Welch Foundation (Grant I-1568) and National Insti-tutes of Health Grant HL102481. To whom correspondence should beaddressed: University of Texas Southwestern Medical Center, 5323 HarryHines Blvd., Dallas, TX 75390. Tel.: 214-648-6477; Fax: 214-648-0320; E-mail:richard.bruick@utsouthwestern.edu.

5 The abbreviations used are: IRP2, iron regulatory protein 2; FBXL5, F-box andleucine-rich repeat protein 5; Hr, hemerythrin; FAC, ferric ammonium cit-rate; DFO, deferoxamine mesylate; SCF, Skp1/Cul1/F-box.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 10, pp. 7357–7365, March 2, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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coordinated by the imidazole side chains from three histidines,whereas the other iron (Fe2) is coordinated by two histidines.Typically, a glutamate and an aspartate provide carboxylategroups that bridge Fe1 and Fe2 (14). In the deoxy state, the ironatoms are also bridged by a �-hydroxo species, ostensiblyderived from the solvent. These hemerythrins often reversiblybind dioxygen to Fe2 with electron delocalization from bothirons to the oxygen to form the oxy-state. A proton is also trans-ferred from the bridging hydroxo to the resulting peroxo ligand.The hydroperoxo adduct is stabilized by a hydrogen bond to the�-oxo bridge (19, 20). Although not previously identified in anyvertebrate proteins, the putative ligand binding properties ofthe Hr domain make it an attractive candidate for a centraliron- and oxygen-sensing module.Herewe report the crystal structure of the isolated FBXL5Hr

domain and characterization of key residues within thisdomain. These data reveal atypical features that allow FBXL5 tofunction via its N-terminal Hr domain as a ligand-dependentregulatory switch in the maintenance of iron homeostasis.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The N terminus ofHomo sapiens FBXL5 (residues 1–161) was cloned into thepGST-parallel1 vector and expressed in Escherichia coli BL21(DE3) cells grown inmedium supplemented with 100 �M ferricammonium citrate (FAC). Soluble FBXL5 Hr protein was puri-fied using glutathione agarose (GE Healthcare). The GSTfusion tag was cleaved upon incubation with tobacco etch virusprotease for 6 h at 24 °C, and the liberated Hr domains werepurified by anion exchange chromatography (HiTrap Q, GEHealthcare). Iron-loaded Hr was separated from apoHr by anadditional anion exchange (Mono Q, GE Healthcare) purifica-tion step. Purified FBXL5 Hr used to generate Native form 1crystals was exchanged into buffer composed of 24 mM Tris-HCl pH8.0, 50mMNaCl, 5mM �-mercaptoethanol, and 0.5mM

tris(2-carboxyethyl) phosphine using a Centricon (Millipore 5kDa molecular weight cut off) and passed through a 0.22-�mfilter.Crystallization and X-ray Diffraction Data Collection—

Crystals of FBXL5 Hr (Native 1) were grown using the hangingdrop vapor diffusionmethod from 1:1 (v/v) mixtures of protein(5 mg/ml) and reservoir solution (10% (w/v) PEG 6000, 0.1 M

HEPES pH 6.5). Plate-like crystals appeared after 1 day at 20 °Cand grew to their maximal extent (0.2 � 0.5 � 0.1 mm) by 2–3days. Cryoprotection was performed by stepwise transfer of thecrystals to a final solution of 25% (v/v) ethylene glycol, 10% PEG6000, and 0.1 M HEPES, pH 6.5, followed by flash-cooling inliquid nitrogen.Crystals of the second form of FBXL5 Hr (Native 2) were

obtained using an identical purification scheme as Native 1,except that recombinant protein was exchanged into bufferlacking reducing agents and allowed to incubate at 4 °C for 24 h.Native 2 crystals were grown using the hanging drop vapordiffusion method from 1:1 (v/v) mixtures of protein (5 mg/ml)and reservoir solution (0.1 M HEPES, pH 7.4, 25% (w/v) PEG3350). Plate-like crystals appeared after 1 day at 20 °C and grewto their maximal extent by 2–3 days. Cryoprotection was per-formed by stepwise transfer of the crystals to a final solution of

0.1 M HEPES, pH 7.4, 25% PEG 3350, and 20% ethylene glycolprior to flash-cooling using liquid nitrogen. Native 2 crystalsexhibited the symmetry of space group P21 with unit cellparameters of a� 76.3Å, b� 54.4Å, c� 78.2Å, and� � 90.02°and contained four molecules of FBXL5 Hr per asymmetricunit. Native 2 crystals displayed moderate anisotropy and P222pseudosymmetry, and diffracted to a dmin of 2.10 Å whenexposed to synchrotron radiation.Crystals of the third form of FBXL5 Hr (Native 3) were

obtained using an identical purification scheme as Native 1except that recombinant protein was exchanged into buffercontaining 2 M equivalent Na2S2O4. Crystals were generated inan anaerobic chamber (Coy Laboratory Products Inc.) using thehanging drop vapor diffusionmethod from1:1 (v/v)mixtures ofprotein (5 mg/ml) and reservoir solution (12% (w/v) PEG 6000,0.1 M HEPES, pH 6.5). Prior to usage, all reagents were purgedwith nitrogen gas for 30 min followed by an overnight incuba-tion in an anaerobic chamber. Plate-like crystals appeared after1 day at room temperature and grew to their maximal extent by2–3 days. Cryoprotectionwas performed by stepwise anaerobictransfer of the crystals to a final solution of 30% (v/v) ethyleneglycol, 12% PEG 6000, and 0.1 M HEPES, pH 6.5, followed byflash-cooling in liquid nitrogen. Native 3 crystals exhibited thesymmetry of space group C2 with unit cell parameters of a �60.4Å, b� 76.0Å, c� 77.4Å, and� � 112.1° and contained twomolecules of FBXL5 Hr per asymmetric unit. Native 3 crystalsdiffracted isotropically to a dmin of 2.20 Å when exposed tosynchrotron radiation. Data were indexed, integrated, andscaled using the HKL-3000 program package (21). Data collec-tion statistics are provided in supplemental Table S1.Structure Determination and Refinement—Phases for Native

1 crystals of FBXL5 Hr were obtained from a two-wavelengthanomalous dispersion experiment using data to a resolution of2.5 Å, collected near the iron absorption edge. Four iron andtwo sulfur sites were located using the program SHELXD (22);this represented two single-occupancy iron sites and onemethionine sulfur site per FBXL5 Hr monomer. Phases wererefined with the programMLPHARE (23), resulting in an over-all figure ofmerit of 0.59 for data between 44.7 and 2.5Å. Phaseswere further improved by density modification and 2-fold non-crystallographic averagingwith the programDM(24), resultingin a figure of merit of 0.85. An initial model containing about86% of all residues was automatically generated by alternatingcycles of the programs ARP/warp (25). Additional residuesweremanuallymodeled in the programO (26). Refinement wasperformed with native data to a resolution of 1.85 Å using theprogram PHENIX (27), with a random 5.1% of all data set asideforRfree calculations. All atoms in the proteinwere restrained ina similar manner, using restraints provided by the PHENIXrefinement program. No explicit metal ligand restraints wereadded.Phases for the Native 2 and Native 3 crystals of FBXL5 Hr

were obtained via molecular replacement in the programPHASER (28) using the coordinates of Native 1 FBXL5 Hr as asearch model. Model building and refinement were performedas described above. The current model for Native 2 crystalscontains four FBXL5 Hr monomers; included are residues5–80 and 83–159 in molecule A, residues 5–73, 77–80, and

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83–159 in molecules B and C, residues 5–73, 77–80, and84–160 in molecule D, and 202 waters. The Rwork is 0.197, andthe Rfree is 0.270. A Ramachandran plot generated with Mol-probity indicated that 98.6% of all protein residues are in themost favored regions, with the remaining 1.4% in additionallyallowed regions. The current model for Native 3 crystals con-tains two FBXL5 Hr monomers; included are residues 4–80and 83–159 in molecule A, residues 4–80 and 83–160 in mol-ecule B, and 46 waters. The Rwork is 0.177, and the Rfree is 0.223.A Ramachandran plot generated with Molprobity indicatedthat 98.7% of all protein residues are in the most favoredregions, with the remaining 1.3% in additionally allowedregions. Phasing and model refinement statistics are providedin supplemental Table S1.EPR Spectroscopy—X-band EPR spectra were recorded using

a Bruker Elexsys E-500 spectrometer equipped with an OxfordInstruments ESR-10 liquid heliumcryostat, under the followingconditions unless otherwise indicated: temperature 10 K, mod-ulation amplitude 1 millitesla, and 5-milliwatt microwavepower, with 200–500 �M FBXL5 Hr as estimated by a calcu-lated molar extinction coefficient of 19.3 mM�1 cm�1 at 280nm.Reduction of the FBXL5hemerythrin domainwas achievedthrough the anaerobic addition of 3 molar equivalents ofNa2S2O4 in the presence of 20 �M methyl viologen in a CoyLaboratory Products anaerobic chamber. The samples weretransferred into sealed quartz EPR tubes and frozen in liquidN2. EPR quantification was performed through comparisonwith a 1 mM copper perchlorate spin standard under nonsatu-rating conditions. Determinations of the exchange couplingconstants of mixed valent FBXL5 Hr were conducted asdescribed previously (29).Stopped-flow Kinetics—Experiments were performed using

an Applied Photophysics Ltd. SX.18.MV stopped-flow spectro-photometer. Following reduction, excess Na2S2O4 and methylviologenwere removed using a PD-10 desalting column (Amer-sham Biosciences) before loading into an anaerobic stopped-flow syringe. Reduced FBXL5 Hr (200 �M) was rapidly mixedwith O2-saturated buffer at 4 °C (�1.8 mM). The rate of FBXL5oxidation at 340 nm under pseudo first order conditions was fitto a summed exponential expression using Pro-K software(Applied Photophysics).Cell Culture and Reagents—All cell lines were grown in Dul-

becco’s modified high glucose Eagle’s medium (HyClone) sup-plemented with 10% fetal bovine serum (Atlanta Biologicals).Plasmid DNA and siRNA transient transfections were per-formed as described previously (12). Low O2 experiments wereperformed in a hypoxic incubator (Coy Laboratory Products)containing 1% O2 and 5% CO2 with the balance being N2. HEK293 cells stably expressing the 3�FLAG-Hr-HAconstructweregenerated over two rounds of clonal selection in the presence of400 �g/ml G418 (Research Products Inc.). Ts-20 BALB/c 3T3cells were kindly provided by H. Ozer.Plasmids—Wild-type, H57A, and E61A human FBXL5 were

cloned into the pCI FLAG vector (kindly provided by X.Wang)as described previously (12). The FBXL5 H15A construct wasgenerated using a triple ligation approach. A 5� DNA fragmentcontaining the H15A mutation was purchased from IDTTechnologies. This fragment was amplified using the oligo-

nucleotide 5�-GGAGAGATCTCAGACCATTTATAATGTA-CATTCTGACAATAAACTC and a previously described WTFBXL5 forward primer (12). The remaining 3� fragment wasamplified from FBXL5 plasmid DNA using the oligonucleotide5�-GGAGAGATCTCAGACCATTGCAGCTGCACATGCT-GACAATAAACTCTCCGAGATGCTT and a previously de-scribed WT FBXL5 reverse primer (12). The resulting frag-ments were ligated together via an artificially inserted BglII siteand inserted into the pCI FLAG vector at the BamHI and SalIsites. The oligonucleotide 5�-GAGGGTCGACTCAAGCGTA-ATCTGGAACATCGTATGGGTACTGAGAGCAGTGTT-GTGC was used to incorporate a C-terminal HA tag into theFBXL5 1–161 pCI FLAG plasmid. The remaining FBXL5mutant and deletion constructs were generated either by stand-ardmethods or by the PCR overlap extensionmethod using theoligonucleotides as listed in supplemental Table S4. For con-structs containing a V5 epitope tag, the pcDNA 3.1 vector(Invitrogen) was used, whereas constructs containing a FLAGepitope were generated in the pCI vector.Immunoblot Analysis—Samples were resuspended in SDS

sample buffer, and proteins were resolved by SDS-PAGE.Mouse monoclonal antibodies were obtained as follows:�-FLAG (Sigma, catalog number F3165),�-V5 (Invitrogen, cat-alog number R960-25), �-tubulin (Sigma, catalog numberT6199), and �-ubiquitin (Santa Cruz Biotechnology, catalognumber sc-8017). Quantitation of immunoblots for Figs. 3 and4, E and F, and supplemental Fig. S4, B and C was performedusing an Amersham Biosciences ImageScanner (PowerlookUDS 1120) and analyzed using the GE Healthcare ImageQuantsoftware (version 5.2). Quantitation of immunoblots for Fig. 4,C and D, was performed using a Kodak Image Station (4000RPro) and analyzed using Carestream molecular imaging soft-ware (version 5.0.2.30). Quantitative data of immunoblots areshown in supplemental Table S5.Ubiquitination Assays—HEK 293T cells stably expressing an

N-terminal 3�FLAG-tagged FBXL5 construct (12) weretreatedwith orwithout 30�MMG132 (BostonBiochem) for 1 hfollowed by the addition of 50 �M FAC (Sigma) or 50 �M defer-oxamine mesylate (DFO; Sigma). Cells were then incubated for6 h. Cell extracts were prepared by adding lysis buffer contain-ing 50mMTris-HCl, pH 8, 150mMNaCl, 1% Triton X-100, 250�M phenylmethylsulfonyl fluoride (Sigma), 1� protease inhib-itor mixture (Sigma), and 10 mM N-ethylmaleimide. Lysateswere incubated 20min at 4 °C, and cysteine (0.1% final concen-tration) was added to neutralize theN-ethylmaleimide. Lysateswere clarified by centrifugation at 17,000 � g for 20 min, andprotein concentration was determined by the Bradford assay(Bio-Rad). Lysates (�1mg) were incubated with 12 �l of FLAGM2 resin (Sigma) overnight to immunoprecipitate FBXL5.Resin was washed three times with lysis buffer, and immuno-precipitated protein was eluted using FLAG peptide. Proteinswere resolved by SDS-PAGE and analyzed by immunoblotting.The ubiquitination assay of the FBXL5 hemerythrin domainwas carried out as described usingHEK293 cells stably express-ing the N-terminal 3�FLAG- and C-terminal HA-taggedFBXL5 hemerythrin domain construct (HEK-Hr). To suppressendogenous FBXL5 expression, HEK-Hr cells were transfectedwith a FBXL5 siRNA (Dharmacon, catalog number D-012424-

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04) and a non-targeting siRNA (Dharmacon, catalog numberD-001210-01) for 48 h.

RESULTS

N Terminus of FBXL5 Adopts Hemerythrin Fold—Recombi-nant FBXL5 Hr was purified under ambient oxygen conditions(supplemental Fig. S1, A and B). The EPR spectrum of the as-isolated Hr (Fig. 1, spectrum 1) shows an S � 1/2 signal withg-values of 1.95, 1.80, and 1.67 (Fig. 1, inset), typical of diironenzymes in the mixed valent state in which antiferromagneticcoupling of high spin FeIII (S � 5/2) and FeII (S � 2) can beobserved (29–31). Reduction with excess sodium dithioniteresults in the disappearance of this signal, and no additionalEPR signal can be observed in either perpendicular (Fig. 1, spec-trum 2) or parallel modes (data not shown), consistent withformation of the diferrous state. Spin quantification shows thatthe EPR-active mixed redox state represents only a minor(5–10%) fraction of the total protein. Themajority of the as-iso-lated FBXL5 Hr likely resides in the EPR-silent (S � 0) diferricresting state based on the intense optical feature at 340 nm(supplemental Fig. S1C) that originates fromanoxo to ferric ioncharge-transfer transition.Crystals of the FBXL5Hrwere generated under ambient oxy-

gen conditions. An x-ray fluorescence scan of these crystalsdisplayed a maximum at an incident energy of 7.12 keV. Thisenergy closely corresponds to the absorption edge of iron (32),implying that this metal is bound to the protein. These Native 1crystals exhibited the symmetry of space groupC2with unit cellparameters of a� 60.1Å, b� 75.8Å, c� 78.7Å, and� � 111.8°and contained two molecules of FBXL5 Hr per asymmetricunit. Native 1 crystals diffracted isotropically to a dmin of 1.85 Åwhen exposed to synchrotron radiation (Native form 1, supple-mental Table S1). The current model for the Native 1 crystalscontains two FBXL5Hrmonomers and includes residues 4–74,77–80, and 84–159 from molecule A and residues 4–74,

77–80, and 84–160 from molecule B along with 168 waters.The Rwork is 0.170, and the Rfree is 0.211. A Ramachandran plotgenerated with Molprobity (33) indicated that 98.3% of all pro-tein residues are in themost favored regionswith the remaining1.7% in additionally allowed regions.Although the FBXL5 Hr up-down �-helical bundle featuring

a diiron center is consistent with the tertiary structure of pre-viously characterized hemerythrins (supplemental Fig. S2A),there are several unusual features present in thisHr, including afifth helix packed against the apex of the canonical bundle (Fig.2A). Overall, the FBXL5 Hr has a similar diameter (�20 Å) butgreater length (58 Å) relative to other hemerythrin structures(�40–50Å) (34, 35) and ismost structurally homologous to theoxygen-responsiveHr from the bacterial DcrHprotein (supple-mental Fig. S3). In canonical Hr structures, each of the iron-ligating residues is positioned within an �-helix (14). However,in the FBXL5Hr, helix�3 is relatively short and does not extendthrough His-80, one of the seven residues comprising the pri-mary iron coordination shell. Instead, this residue exists in asmall ordered region that is in a partially disordered loop pre-ceding helix �3 (Fig. 2 and supplemental Fig. S3A). The coordi-nation geometry of the FBXL5 Hr diiron site also differs fromthose of canonical hemerythrins. As noted in supplementalTable S2, the Fe-Fe distance is shorter in the FBXL5 Hr (3.12 Åversus�3.34–3.41Å) than other hemerythrins (14, 35), and theFBXL5Hr Fe-O�-Fe angle is more acute (98.1°) than those pre-viously observed in invertebrate (116.7°) and bacterial (132.4°)hemerythrins (14, 35). Moreover, the core iron atoms are posi-tioned closer to the apex of the bundle in the FBXL5 Hr (sup-plemental Fig. S3B).IronBinding byHemerythrinDomain Is Required for Stability

of FBXL5—Examination of the amino acid side chains respon-sible for coordinating iron (Fig. 2, B and C) reveals differencesbetween the FBXL5 diiron center and the consensus observedin the majority of hemerythrins. In place of an aspartate andglutamate to bridge the diiron center, the FBXL5 Hr employstwo glutamates (Glu-61 and Glu-130). As most Hr domainsstudied to date employ five coordinating histidines, the mostsignificant variation in the FBXL5 Hr diiron center is the pres-ence of an additional glutamate (Glu-58) in lieu of a third histi-dine binding Fe1. Interestingly, the distance between O�2 ofGlu-58 and the bridging oxygen is �2.8 Å (supplemental TableS2) with an O�-O�2-C� angle of �106°. This geometry is con-sistent with Glu-58 forming a hydrogen bond with a �-oxygenspecies (supplemental Fig. S2B).To confirm the critical roles of the observed iron-ligating

residues to iron-dependent regulation of FBXL5, each residuewas individually mutated to an alanine. The resulting expres-sion constructs were transfected into HEK 293T cells prior totreatment with the iron chelator DFO or the iron species FAC.In contrast to wild-type (WT) FBXL5, which preferentiallyaccumulates under iron-replete conditions, all seven mutantproteins displayed constitutively low accumulation levels (Fig.3A) that could be rescued by the addition of the proteasomeinhibitor MG132 (supplemental Fig. S4A). In addition to thesedirect iron-binding ligands, the FBXL5 Hr structure indicatedthat residues Asn-62, Glu-131, and His-158 form hydrogenbondswith the primary coordination shell residuesHis-80,His-

FIGURE 1. EPR spectra of FBXL5 Hr domain. Spectra 1 and 2, the 20 K EPRspectrum of the Hr domain following purification under ambient oxygen con-ditions (spectrum 1) and upon the addition of excess reducing agent (spec-trum 2). Comparison with a canonical Hr from P. gouldii is shown in the inset.

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15, and His-57, respectively (Fig. 2B). We postulated that thesesecondary shell interactions might be crucial for the stable for-mation of the diiron center by bringing the imidazole moietiesof residues His-80, His-15, and His-57 into their optimal con-formations to coordinate iron. Constructs containing muta-tions to secondary shell residues (N62A, E131A, and H158A)were transiently transfected intoHEK 293T cells, and they, too,displayed lowered protein levels under both iron-replete andiron-deficient conditions (supplemental Fig. S4B). Loss of theadditional fifth helix (�143–161), found in few other hemeryth-rin-like structures (PDB ID 2P0N), also led to constitutivedestabilization of FBXL5 (supplemental Fig. S4C). This resultwas expected, given the importance of the secondary coordina-tion shell residue His-158 within helix �5.In the FBXL5 Hr domain, Fe1 is ligated by two histidines

(His-126 and His-80) and a glutamate (Glu-58) that appears tomake a hydrogen bond with the bridging, solvent-derived oxy-gen atom. To directly interrogate whether this unique ligandenvironment alters the electronics of the diiron site, we deter-mined the exchange coupling constant J of the mixed valentFBXL5 Hr to be 120 cm�1 (supplemental Fig. S5). This largevalue is suggestive of an oxo bridge, rather than a hydroxobridge (J � 20 cm�1), as observed for mixed valent forms of thecanonical Hr (30, 31) (supplemental Table S3). Hence, unlikeHr and other diiron enzymes, in which reduction from thediferric to the mixed valent state is accompanied by protona-tion of the single atom bridge, the bridge of FBXL5 is likelyretained as an oxo ligand.

To determine whether this unusual arrangement wasrequired for Hr function, FBXL5 Glu-58 was mutated to a his-tidine (E58H). As observed for the E58A variant, E58H FBXL5was constitutively unstable following both FAC andDFO treat-ment (Fig. 3B). Likewise, when residue Glu-58 was mutated toaspartate (E58D) or glutamine (E58Q), FBXL5was again unsta-ble and unresponsive to iron (Fig. 2C). The failure of histidineor aspartate to functionally substitute for Glu-58 could be dueto steric constraints. Modeling indicates that there is anuncommon, high energy His rotamer that could place the N�1atom within 2.8 Å of the iron. However, a more common, lowenergy rotamer position for His-58 would place the N�2 atom3.7 Å from Fe1, too far away to function as an iron ligand.Similarly, aspartate at position 58 would place a carboxylateoxygen �2.6 Å from Fe1, which is still rather far for an ironligand, whereas the second carboxylate oxygen is also too farfrom the �-hydroxo atom to function as a hydrogen bondacceptor.Moreover, a glutamine at position 58 also fails to bindiron (data not shown) and restore proper iron responsiveness(Fig. 3B), although its carboxamide oxygen can on rare occa-sions ligate iron. Together, these data suggest that FBXL5induction in response to increased iron bioavailability requiresan intact Hr domain capable of assembling a diiron center withunusual ligands.FBXL5 Is Polyubiquitinated and Degraded by Proteasome

under Iron-deplete Conditions—Destabilization of FBXL5upon iron depletion is dependent on the proteasome (12). Todetermine whether the proteasome sensitivity of FBXL5 is

FIGURE 2. Crystal structure of FBXL5 hemerythrin domain. A, ribbon representation of the hemerythrin domain from H. sapiens FBXL5 (residues 5–159; PDBcode 3V5X). Helices are shown in green, and loops are in shown in red. The additional fifth C-terminal helix found is shown in yellow. The dotted lines representthe disordered residues 75–76 and 81– 83. B, the first coordination sphere iron ligands of the FBXL5 Hr diiron center are shown as green sticks, and the conservedsecond coordination sphere iron ligands are shown as pink sticks. Iron ligands and hydrogen bonds are shown as dotted black lines. C, diiron center of FBXL5 HrNative 1, monomer A with superimposed Fo � Fc simulated annealing omit electron density map (gray, contoured at 3.0 �) calculated for the bridging oxygen(red) and the side chains (green) of the first coordination sphere (atoms beyond the C-� for the amino acids were omitted). An anomalous difference Fourierelectron density map (red, contoured at 15.0 �) is shown for the iron atoms (cyan).

FIGURE 3. Regulation of FBXL5 is dependent on assembly of diiron center within hemerythrin domain. Eight hours after transfection, HEK 293T cells weretreated with either 100 �M DFO or 100 �M FAC for 16 h, and FBXL5 accumulation was assessed by immunoblot analysis. A, analysis of mutations to residuescomprising the primary iron coordination shell. B, analysis of FBXL5 Glu-58 mutations.

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accompanied by polyubiquitination in an iron-dependent fash-ion, HEK 293T cells stably expressing FBXL5-FLAG weretreated with either FAC or DFO in the absence or presence ofMG132, and FBXL5-FLAG was immunoprecipitated. Asshown in Fig. 4A, FBXL5 is polyubiquitinated under iron-defi-cient conditions. However, when iron is plentiful, the extent ofthe laddering characteristic of heterogeneous polyubiquitina-tion is diminished (Fig. 4A). These data are consistent withbioavailable iron levels regulating FBXL5 via its polyubiquitina-tion status.Similar to full-length FBXL5, the isolated FBXL5 Hr domain

is preferentially polyubiquitinated under iron-deficient condi-tions (Fig. 4B). To further confirm that ubiquitination isrequired for FBXL5 Hr degradation, a murine ts-20 BALB/c3T3 fibroblast cell line featuring a temperature-sensitive E1variant (36, 37) was transfected with a FLAG-Hr-HA construct.At the E1-permissive temperature (35 °C), FLAG-Hr-HA levelsdiminish upon a shift from iron-replete conditions to iron-de-plete conditions (Fig. 4C). At the restrictive temperature(39 °C), loss of E1 activity globally compromises polyubiquiti-nation and degradation of substrates by the ubiquitin-protea-some system (37, 38). Under these conditions, FLAG-Hr-HAprotein accumulation levels remain high, even upon the addi-tion of DFO (Fig. 4C). Likewise, a functional ubiquitin-protea-some system is required for FBXL5 Hr degradation under lowO2 conditions (Fig. 4D).

As the F-box subunits of Skp1/Cul1/F-box (SCF) E3 ubiqui-tin ligase complexes can be subjected to autoubiquitination(39), we suspected that SCFFBXL5might itself be the responsibleHrE3 ligase.However, the isolatedHr lacks a F-box domain andthus cannot assemble into an SCF E3 ligase complex, arguingagainst an autoubiquitinationmodel. To exclude the possibility

that SCFFBXL5 could ubiquitinate the FBXL5 Hr domain intrans, endogenous FBXL5 was depleted from HEK-Hr cellsusing an siRNA selectively targeting the full-length FBXL5mRNA. Although FBXL5 knockdownwas sufficient to stabilizeIRP2, no effect on accumulation or ubiquitination status of theisolated Hr domain was observed (Fig. 4B). These data suggestthat another, hitherto unidentified E3 ubiquitin ligase activity isrequired for FBXL5 regulation conferred through its Hrdomain.To map an element within the FBXL5 Hr domain that is

required for targeting the protein for degradation, a series ofprogressively longer FBXL5 N-terminal truncation constructswas generated. Because such large truncationswere expected tocompromise assembly of the diiron center, and consequentlyHr integrity, it was postulated that all deletions would produceiron-insensitive and constitutively unstable products.Although the�1–33,�1–59, and�1–76 proteins were predict-ably unstable, the �1–81 and �1–100 proteins accumulated tohigh levels following both FAC and DFO treatment (Fig. 3C),suggesting that there is a regulatory sequence required forFBXL5 degradation located C-terminal to residue Ile-76. Tobetter delineate this sequence, we generated a series of FBXL5constructs containing adjacent 5-amino acid deletions in theregion spanning residues 67–91. Interestingly, deletion of resi-dues 77–81 was sufficient to confer a substantial constitutiveincrease in FBXL5 accumulation (Fig. 4E) despite the fact thatloss of His-80 should preclude any iron binding. We proposethat these residues are a required part of a regulatory sequencemediating FBXL5 proteasomal degradation. Notably, thissequence resides in the unusual extended loop joining helices�2 and �3 (Fig. 2A and supplemental Fig. S3A).

FIGURE 4. FBXL5 degradation under iron-deplete conditions requires functional ubiquitin-proteasome system and regulatory sequence. A, immuno-blot analysis of immunoprecipitated (IP) FLAG-FBXL5 from stably transfected HEK 293T cells treated with FAC or DFO in the absence or presence of theproteasome inhibitor MG132. B, ubiquitination of FBXL5 Hr is not dependent on FBXL5. HEK 293 cells stably expressing the FBXL5 Hr domain were transfectedwith a negative control siRNA (NT) or siRNA targeting endogenous FBXL5 prior to treatment with FAC or DFO in the absence or presence of MG132. C,immunoblot analysis of FLAG-Hr-HA accumulation in ts-20 BALB/c 3T3 cells incubated with FAC or DFO for 6 h followed by a switch to either E1-permissive(35 °C) or E1-restrictive (39 °C) temperatures for 24 h in medium containing FAC or DFO as indicated. D, immunoblot analysis of FLAG-Hr-HA accumulation ints-20 BALB/c 3T3 cells. Cells were incubated with FAC for 6 h and maintained in 1 or 21% O2 at 35 °C or 39 °C for 24 h. E and F, immunoblot analysis of FBXL5accumulation from expression constructs having increasingly longer N-terminal deletions (E) or short internal deletions (F). FL, full length.

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Oxygen-dependent Regulation of FBXL5—IRP2 degradationis induced by both high iron and high oxygen availability (5, 6)and is proposed to be largely conferred through the effects ofiron and oxygen on FBXL5 (40). Similar to ectopicallyexpressed FBXL5 (12), endogenous FBXL5 accumulation in thepresence of excess iron is attenuated in a proteasome-depen-dent manner when cells are incubated under low O2 condi-tions.6 The isolated FBXL5Hr domain also directly responds toO2 (Fig. 4D), suggesting that oxygen availability affects the sta-bility of FBXL5 via polyubiquitination and proteasomal degra-dation mediated by its Hr domain.The majority of previously characterized hemerythrins have

been shown to reversibly bind oxygen at the diiron center (34,41, 42). Although FBXL5 and its Hr domain are clearly regu-lated in an oxygen-dependent manner, no ordered electrondensity corresponding to O2 was observed at the diiron centerof the FBXL5 Hr crystal structure (Fig. 2B). Interestingly, nei-ther FBXL5 Hr structures derived from crystals grown in theabsence of reducing agent (Native form 2, supplemental TableS1) nor structures derived under anoxic conditions in the pres-ence of excess dithionite reductant (Native form 3, supplemen-tal Table S1) were appreciably different (supplemental Fig.S6A).Closer examination of the FBXL5 Hr diiron center reveals

that molecular oxygen would not be able to bind to the diironcenter in these structures.When comparing FBXL5Hr and theO2-binding DcrHHr, the spatial conservation of the iron-ligat-ing residues with respect to the diiron center is remarkablysimilar, although there are significant spatial variations in thehydrophobic amino acids lining the putative O2-binding pock-ets (Fig. 5A). In the FBXL5Hr, Phe-123 is positioned toward thecenter of the helical bundle and in close proximity (�4Å) to thebridging�-oxygen atom.Met-127 is also positioned closely (�4Å) to both the bridging oxygen atom and Fe2. In the DcrH Hrcrystal structure (35), Trp-114, the corresponding hydrophobicresidue to Phe-123, is near the periphery of the hydrophobiccore adjacent to Fe1. The analogous residue to Met-127 in theDcrH Hr, Leu-115, is more distal (�6 Å) to both Fe2 and thebridging oxygen. Consequently, the binding pocket in theDcrHHr hasmuch larger dimensions than the FBXL5Hr site and canaccommodate an O2 molecule. In fact, there is not even suffi-cient room to model a molecule of water solvent in the Fe2coordination site. Thus, substantial rearrangements to theFBXL5 Hr core would be required for ligand binding.Rapid mixing of diferrous FBXL5 with saturating dioxygen

results in the rapid reappearance (t1⁄2�250 ms) of the Fe-oxocharge transfer band at 340 nm with no other transient inter-mediate accumulating prior to reoxidation to the diferric rest-ing state (Fig. 5B). In particular, no oxygenated form of thedomain, such as an end-on hydroperoxo moiety observed inPhascolopsis gouldii Hr (� �2000 M�1cm�1 at 500 nm) or a�-1,2-peroxo adduct (typically observable at �700 nm), can beobserved following dioxygen addition. Thus, although the oxi-dation state of the FBXL5Hr diiron center responds toO2 avail-ability, its degron accessibility is not likely to be regulated by

conformational changes stemming from reversible binding ofO2.

DISCUSSION

Here we employed structural and molecular approaches tocharacterize the Hr domain that mediates regulation of the E3ligase subunit, FBLX5. X-ray crystallography demonstratedthat the isolated FBXL5 N terminus adopts a hemerythrin-like�-helical bundle fold stabilized by a diiron center. Nevertheless,this mammalian Hr contains several unique attributes thatcould contribute to its switch-like properties including 1) arequired fifth helix contributing a residue in the secondary ironcoordination shell, 2) a noncanonical primary iron ligand thatalso makes an apparent hydrogen bond contact with the bridg-ing solvent-derived oxygen atom, 3) crowded side-chain pack-ing within the presumptive O2-binding site, and 4) a truncatedhelix �3 preceded by several disordered amino acids and anextended loop that contains an important regulatory element.Under iron- and oxygen-replete conditions, both FBXL5 and

its isolated Hr are resistant to polyubiquitination and protea-6 S. Chollangi, J. W. Thompson, J. C. Ruiz, and R. K. Bruick, manuscript in

preparation.

FIGURE 5. FBXL5 Hr domain does not appear to bind oxygen ligand. A,comparison of the FBXL5 and DcrH diiron centers. Shown in stereo is a super-position of the bound iron atoms (dark blue balls), bridging oxygen (dark redball), and first coordination sphere iron ligands (green) of FBXL5 Hr (PDB code3V5Y) with corresponding atoms (pale balls) and ligands (yellow) of deoxy-DcrH Hr (PDB code 2AWC). B, stopped-flow kinetics of reduced anaerobicFBXL5 Hr rapidly mixed with O2 saturated buffer. Fitting of the reoxidation ofthe Hr domain at 340 nm required three summed exponentials to adequatelyaccount for the observed data (inset), indicating a complex oxidation process.Abs, absorbance.

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somal degradation. Compromising formation of the diiron cen-ter, either through chelation of bioavailable iron or bymutationof any residue within the primary or secondary iron coordina-tion spheres, leads to increased degradation of FBXL5. Therequirement for E1 activity suggests that ubiquitination isrequired for FBXL5 degradation. Although the responsible E3ligase has not yet been identified, residues 77–81 appear to actas part of a functional degron as deletion of this region stabilizesthe protein, despite the inability of the Hr domain to bind ironupon removal of His-80.We propose that in the absence of ironbinding, this regulatory sequence becomes fully accessible, pro-moting a large induction in the extent of polyubiquitination andproteasomal degradation.Despite our efforts to crystallize both oxidized and fully

reduced forms of FBXL5 Hr, the resulting structures do notdisplay statistically significant differences in bond distances orangles of the diiron centers. Given the observed Fe1-Fe2 dis-tances (supplemental Table S2) and preparation conditions, itis most likely that these structures all represent a mixed valentor diferric state. However, the observation of a putative hydro-gen bond between Glu-58 and the bridging oxygen atom is dif-ficult to reconcile if the FBXL5 is present in the met or semi-met state in the crystal. Under the working buffer conditions(pH 6.5 and 7.4), the probability that Glu-58 is protonated andserving as a hydrogen bond donor to a �-oxo bridge is small,particularly as it is serving as an iron ligand. Only the reduceddiferrous Hr could provide a requisite H-bond donating �-hy-droxyl group as both the diferric and themixed valent semi-metstates feature a �-oxo bridge. As crystallization could trap aminor reduced species, or the oxidation state of the Hr couldchange during crystal manipulations (15), we cannot unambig-uously assign the oxidation states of our crystals.Among the Hr domain structures solved to date, the FBXL5

Hr is most structurally homologous to the bacterial DcrH Hr,anO2 sensorwithin a bacterial chemotaxis protein (35, 42). TheDcrH Hr contains a putative substrate channel that is thoughtto facilitate diffusion ofO2 to the diiron center. Oxygen bindingto the DcrH Hr causes a subtle conformational change in anN-terminal loop, which may drive further conformationalchanges within the full-length DcrH protein to govern its aer-otactic signaling function (35).Instead of a channel, there is a large solvent-accessible sur-

face near Fe1 of the FBXL5 Hr diiron center facilitating oxygenexposure (supplemental Fig. S7). However, FBXL5 does nothave an analogousN-terminal loop and, based on the structurespresented here, it would have to undergo a substantial confor-mational change displacing residues Phe-123 and Met-127 toaccommodate an O2 ligand at the Fe2 site. Another potentialhindrance to O2 binding the diiron center is residue Glu-58,which appears to engage the bridging oxygen in a hydrogenbond interaction.Moreover, no evidence for transient O2 bind-ing was observed during reoxidation experiments or in thestructures themselves, although oxygen was present through-out two of the three protein and crystal preparations. Alterna-tively, the O2 responsiveness observed for the isolated Hrdomain in vitro and in cell culture studies could originate fromelectron transfer through outer sphere amino acid ligands.

Overall, the data presented here are consistent with a modelin which the FBXL5 N-terminal Hr domain senses both ironand oxygen to regulate FBXL5 stability.When intracellular ironlevels are abundant, the Hr domain binds iron and adopts aconformation in which a degron containing residues 77–81 issequestered. Notably, in these crystal structures, the loop fromresidues 74–83 is not well ordered as evidenced by the highatomic displacement parameters for this region (supplementalFig. S6B). This feature is unique to the FBXL5 Hr domain andoverlaps a key regulatory element required for degradation. It istempting to hypothesize that low oxygen conditions facilitate aphysiologically relevant increase in accessibility of helix �3,predisposing the FBXL5 Hr domain to enhanced degradation.

Acknowledgments—The structure shown in this report is derived fromwork performed on beamlines 19-BMand 19-ID at ArgonneNationalLaboratory, Structural Biology Center at the Advanced PhotonSource. UChicago Argonne, LLC, operates Argonne for the UnitedStates Department of Energy, Office of Biological and EnvironmentalResearch under Contract DE-AC02-06CH11357. This investigationwas conducted in a facility constructed with support from theResearch Facilities Improvement Program (Grant C06 RR 15437-01)from the National Center for Research Resources, National Institutesof Health.

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BruickBrautigam, Thomas M. Makris, John D. Lipscomb, Diana R. Tomchick and Richard K. Joel W. Thompson, Ameen A. Salahudeen, Srinivas Chollangi, Julio C. Ruiz, Chad A.

Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5)Structural and Molecular Characterization of Iron-sensing Hemerythrin-like

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