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`B OCHEMISTRY
`MIWWlmm
`”Article
`
`Biochemistry 2009, 48, 9569.9581
`DOI: 10.1021/bi901182w
`
`9569
`
`The Yeast iron Regulatory Proteins er3/4 and Fra2 Form Heterodimeric Complexes
`Containing a [2Fe-ZS] Cluster with Cysteinyl and Histidyl Ligation:r
`
`Haoran Li} Daphne T. Mapolelo.§ Nin N. Dingrafounil G. Naik," Nicholas S. Lees} Brian M. Hoffman:L
`Pamela J. Riggs-Gelaseof Boi Hanh l-luynh,I Michael K. Johnson,*‘§ and Caryn E. Outten‘“:
`:Department: ofChemistry and Biochemistry. University o/Sourh Carolina. Columbia, South Carolina 29208,
`’Department of Chemistry and Centerfor Metalloenzyme Studies, University of Georgia, A rhenr, Georgia 30602. "Department of
`Physics, Emory University, Atlanta, Georgia 30322, J'Chemistry Department, Northwestern University, Evanston. llll'nols 60208. and
`Department of Chemistry and Biochemistry, College of Charleston. Charleston, Soul/1 Carolina 29424
`Received July 10, 2009: Revised Manuscrr'pr Received A ugusr 28, 2009
`
`ABSTRACT: The transcription of iron uptake and storage genes in Sacrhammycer ccrevisiae is primarily
`regulated by the transcription factor Aftl. Nucleocytoplasmic shuttling of Aftl
`is dependent upon
`mitochondrial Fe-S cluster blosymhesis via a signaling pathway that includes the cytosolic monothiol
`glutaredoxins (er3 and er4) and the ROM homologue Fra2. However, the interactions between these
`proteins and the iron-dependent mechanism by which they control Aftl
`localization are unclear. To
`reconstitute and characterize components of this signaling pathway in virro, we have overexpressed yeast
`Fra2 and er3/4 in Escherichia coli. We have shown that coexpression of recombinant Fra2 with er3 or
`er4 allows purification of a stable [2Fe-ZS]2+ cluster—containing Fra2-er3 or FraZ-er4 heterodimerlc
`complex. Reconstitution of a [ZR->25] cluster on er3 or er4 without FraZ produces a [2Fe—25]~bridgcd
`homodimer. UV—visible absorption and CD, resonance Raman, EPR, ENDOR, Méssbuuer, and EXAFS
`studies of [2Fe-ZS] er3/4 homodimers and the [2Fe-2S] Fra2—er3/4 heterodimers indicate that inclusion of
`Fra2 in the Gl‘X3/4 Fe-S complex causes a change in the cluster stability and coordination environment.
`Taken together, our analytical, spectroscopic, and mutagenesis data indicate that er3/4 and FraZ form a Fc—
`S—bridged heterodimen'c complex with Fe ligands provided by the active site cysteine ofer3/4, glutathione,
`and a histidine residue. Overall, these results suggest that the ability ofthe Fra2-er3/4 complex to assemble a
`[2Fe-ZS] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast.
`
`Maintenance of optimal iron levels inside the cell is critical for
`all eukaryoles and most prokaryotes, for iron is both essential
`and potentially toxic. As a protein cofaotor, iron can bind directly
`to amino acids, forming mono- or dinuclear iron centers, or it can
`be incorporated with porphyrins or sulfide to form home or Fe—S
`clusters, respectively. However, uncontrolled iron redox chem-
`istry can lead to formation of reactive oxygen species, causing
`extensive cellular and molecular damage that eventually leads to
`cell death (1). Therefore, cells must be able to sense iron levels and
`regulate iron homeostasis accordingly to maintain critical, non-
`toxic levels of this key nutrient.
`The expression of iron uptake and storage genes in the model
`eukaryote Sarcharomyces cereviriae is primarily controlled by the
`Fo—rcsponsive transcription factor Aftl. A01 is located in the
`cytosol under iron—replete conditions and moves to the nucleus
`
`TThis work was supporLed by the NIH (ESl3780. C.E.0.: GM62524,
`M.K.J,; GM47295, B.H.H.; l-IL13531, B.M.H.; P20 RR01646l, P.J.R,
`-G.) and by the Camille and Henry Dreyfus Foundation (Henry Dreyfus
`Teacher-Scholar Award to P.J.R.-G.). Both the National Synchrotron
`Light Source and the Stanford Synchrotron Radiation Laboratory are
`national user facilities supported by the US, Department of Energy.
`Office of Basic Energy Sciences. The SSRL Structural Molecular
`Biology Program is supported by DOE and the NlH-NCRR Biomedical
`Technolo
`Program.
`.
`*To w om correspondence should be addressed, C.E.0.: e-marl,
`caryn,outten@ehem.sc.edu; tel, 803-777-8783; fax. 803-777-9521. M.
`K}: e-mail, johnson@chem.uga.edu; tel, 706-542-9378; fax, 706-542-
`9454.
`
`© 2009 American Chemical Sou'ely
`
`under iron-depleted conditions, where it activates genes involved
`in high-affinity ionic iron uptake, siderophore iron uptake. and
`vacuolar
`iron transport, known collectively as
`the iron
`regulon (2—7). Nucleocytoplasmic shuttling of Aftl in yeast is
`regulated by mitochondrial Fe-S cluster biosynthcsis via a
`signaling pathway that includes the cytosolic monothiol glutar-
`edoxins (er3l and er4), the BolA homologue Fra2 (l~‘e
`repressor of activation—2), and the aminopeplidase P-like protein
`Fral (Fe repressor of activation-l) (8-11). When Fe-S cluster
`biosynthesis is active (i.e., under Fe replete conditions),
`this
`signaling pathway is proposed to induce multimerization ofA01
`in an unknown manner to promote its export from the nu-
`cleus (12), A 29‘CXCm motifis required for Aftl translocation in
`response to iron. but the specific function of these residues is
`unclear (2, I l, 12). Under low iron conditions or upon disruption
`of mitochondrial Fc-S cluster biogenesis, the FrallFroZ/er3/
`er4 pathway is shut off, allowing Aftl to accumulate in the
`nucleus and activate iron uptake systems. Despite the identifica-
`tion of multiple components in this signaling pathway,
`the
`
`‘Abbreviations: er. glutaredoxin: 'I‘rx, thioredoxin; GSH, reduced
`glutathione; GSSG, oxidized glutathione; MALDI—TOF. matrix-as-
`sisted laser desorption ionization-time of flight; CD, circular dichroism;
`EPR. electron paramagnetic resonance; ENDOR. electron-nuclear
`double resonance spectroscopy; EXAFS. extended X-ray absorption
`fine structure: IPTG. isopropyl fi-n-thiogalactoside; NSLS, National
`Synchrotron Light Source; SSRL, Synchrotron Radiation Laboratory:
`CN, coordination number; FT. Fourier transform.
`
`Published on Web 08/28/2009
`
`pubs.acsorg/Biochemistry
`
`
`
`

`

`9570 Biochemistry, Val. 48. Na. 40, 2009
`specific mechanism of iron—dependent regulation of Aftl locali-
`mtion by Fra2 and er3/4 is a key gap in our understanding of
`intracellular iron metabolism.
`Yeast er3 and er4 are members of the monothiol glutar-
`edoxin (er) family, which is found in organisms ranging from
`bacteria to humans. er3 and er4 are highly homologous
`proteins that possess both an N-terminal thioredoxin~ (Trx-) like
`domain and a C‘temtinal Grit-like domain (13). Cytosolic Grid
`and er4 perform redundant functions since deletion of each
`gene singly has little effect on iron regulation, while a grx3A
`grx4o double mutant exhibits constitutive expression of iron
`regulon genes (9, 10). The putative active site in the Grit—like
`domain of er3/4 has a highly conserved CGFS motif that is
`specifically required for interaction with Aftl and regulation ofits
`activity (9). A third CGFS’type monothiol Got in yeast (er5) is
`localized to mitochondria where it plays an essential but
`ill—defined role in mitochondrial Fe-S cluster biogenesis (14, 15).
`The human homologue of the yeast cytosolic monothiol ers
`(11er3, also known as PICOT for PKC—interacting cousin of
`Trx) was initially identified in T lymphocytes where it acts as a
`negative regulator of protein kinase 00 via an unknown mechan-
`ism (16). More recently, mammalian er3 was shown to inhibit
`cardiomyocytc hypertrophy (i.e.. thickening of the heart muscle)
`by binding to the muscle LlM protein (ML?) and blocking the
`stress-responsive. prohypertrophic caicineut‘in-nuclear factor of
`the activated T-txll (NFAT) signaling pathway (I7).
`ers are part of the Trx-fold superfamily and typically
`catalyze thiol—disulfide exchange reactions via monothiol or
`dithiol mechanisms ( I8). The dithiol mechanism requires two Cys
`in the active site (usually in a CPYC motif), with the N-terminal
`Cys forming a mixed disulfide between er and the target
`protein, which is resolved by the second active site Cys. The
`monothiol mechanism requires only the N-terminal Cys in the
`active site and is used for reducing mixed disulfides between GSH
`and target proteins. Although the N-terminal Cys is conserved in
`CGFS-type monothiol er, they lack oxidoreductase activity
`when tested with standard er model substrates (18). Several
`members ofthe monothiol ers family, including yeast er3 and
`er4. were recently shown to form [2Fe—25]~bridged homodimers
`with the CGFS active sites providing two Cys ligands (IO—~21).
`interestingly. two glutathione molecules (GSH or y—glutamyl—
`cysteinyiglycine) provide the other two cluster ligands. GSl-I is an
`abundant lripeptide that serves as the primary intracellular thiol
`redox buffer and a cofactor for glutaredoxins and other anti-
`oxidant enzymes (22). in the three published crystal structures of
`[2Fe-ZSl-bridged dithiol and monothiol er proteins, the GSH
`molecules are covalently linked to the cluster but held in place by
`noncovalent interactions with theGSH binding pocket in the Gut
`protein (2]. 23, 24). GSH seems to play a role in yeast er3/4
`function since GSH binding residues in er3/4 are essential for
`regulation of Aftl activity (9). Based on previous studies. several
`possible functions for monothiol ers in iron metabolism have
`been proposed. ers may act as scaffolds for Fe-S cluster
`assembly. delivery proteins for Fe-S cluster transfer, regulators
`of Fe-S cluster assembly, or Fe-Sdependent sensors that
`relay cellular
`iron status
`to iron-responsive transcription
`factors (19—21). In any case,
`the unusual GSH-ltgalecl Fe-S
`cluster in monothiol Grits directly links iron homeostasis wtth
`Fe-S cluster assembly and thiol redox regulation.
`,
`Several
`lines of evidence suggest that the monothiol ers
`function together with another widely conserved protein family,
`the BolA-lilte proteins (25). 301A was originally identified in
`
`Li et a1.
`
`Article
`
`Escherichia call as a protein that induces a round cell shape when
`overcxpresscd (26). Although BolA—like proteins are found in
`a wide variety of prokaryotes and eukaryotes, their specific
`molecular function is unknown. Genome-wide yeast two-hybrid
`assays have identified a physical interaction between cytosolic
`monothiol ers and BolA-like proteins in both S. cerevisiae and
`Drasaphila malanogaster (27, 28). In addition, proteome—wide
`FLAG— and TAP-tag affinity purification studies in yeast and
`E. cali have shown that BolA-like proteins such as Fra2 copurify
`with monothiol ers (29*31). The physical interaction between
`yeast er3/er4 and the BolA—iike protein Fra2 was recently
`confirmed by inununoprecipitation and split YFP tagging (8).
`Finally, comparative genomic analyses also predict a functional
`interaction between monothiol ers and BolA-like proteins
`since they are neighbors in several prokaryotic genomes (25).
`Furthermore. genes encoding the BolA and monothiol er
`proteins exhibit strong genome cooccurrence since almost all
`organisms that possess a BolA-like protein also have a CGFS
`monothiol er, while organisms that lack a BolA-like protein
`almost always lack a CGFS-type er (25, 32). Taken together,
`these data demonstrate a strong phylogenetic connection be-
`tween the two protein families. However.
`the nature of the
`interaction between CGFS-type ers and BolA—like proteins
`has not been previously determined
`This study is aimed at characterizing the interaction between
`yeast er3/4 and Fra2 in vitro using biochemical, spectroscopic,
`and analytical techniques. We show here that both er3 and
`er4 form [2Fe-ZS]-conlaining heterodimers with Fra2 that have
`similar Fe—S coordination environments. However, the UV—vi—
`sible absorption. CD.
`resonance Raman. EPR. ENDOR,
`Mossbauer, and EXAFS spectra of reconstituted [ZFe-ZS]-
`bridged er3 or er4 homodimers are markedly different from
`[2Fe-ZS] Fra2-Grit3/4 heterodimers,
`indicating differences in
`cluster coordination. Furthermore, we have determined that
`conserved residues required for er3 and er4 Fe signaling
`in vivo are also required for Fe-S complex formation with Fra2
`in vitro. This study thus provides new insight into the molecular
`details of intracellular Fe signaling and establishes the ubiquitous
`monothiol ers and BolAalike proteins as a novel type of Fe-S
`cluster binding regulatory complex.
`
`EXPERIMENTAL PROCEDURES
`Plasmid Construction. Construction of the yeast er3
`E. calf expression vector pET21a~er3 was described pre-
`viously (33), The ORF of yeast er4 were amplified from
`S. cerevisiae genomic DNA by PCR using the primers shown
`in Supporting Information Table l and cloned into the NdeI and
`Ele sites of pETZla (Novagen) to generate pETZla-er4.
`E. cult expression vectors for His—tagged Fral (pETZla-Fral-
`His.) and Fra2 (pET21a-Fra2-Hi56) were kindly provided by
`Jerry Kaplan (University of Utah) (8). The untagged Fra2
`expression vector pETZla»Fra2 was constructed by amplifying
`the Fra2 ORF without the His tag from pETZIa-FraZd-iisé and
`reinserting the gene at the Ndel and Sacl sites in pETZla (see
`printers in Supporting Information Table l). Fral-Hist. (from
`pETZla-Fral-Hise) and untagged Fra2 (from pET21a~Fra2)
`were subcloned into the first and second multiple cloning
`sites (Nail/Sari and Ndel/Kpnl) of the coexpression vector
`pRSFDueM (Novagen), respectively. to generate pRSFDuet~
`l~Fral-Hise/Fra2
`and pRSFDuet-l-Fral. er3 mutants
`were created by sitedirectod mutagenesis of pETZln-er3
`
`‘
`
`(QuikChange kit; Stratagene) using primers listed in Supporting
`Information Table 1. pETZla-er3(Al-121) was created by
`introducing an NdeI restriction site at position 122 in pETZla—
`er3 by site-directed mutagenesis, digesting the plasmid with
`Ndel to remove the coding sequence for amino acids 1—121, and
`religating the plasmid. pET21a-er3(A122-250) were created
`by introducing a stop codon and Hz'ndIII site at position 122,
`digesting the plasmid with HindlIl to remove the coding sequence
`for amino acids 122-250, and religating the plasmid. The cDNA
`for human er2 (herZ) (Open Biosystcms) was PCR amplified
`without the mitochondrial targeting signal (amino acids 41 — 164)
`and subclotied into pET24d (Novagcn) using the Neal and
`EcoRI restriction sites to make pET24d-her2. The sequence
`integrity ofall plasmids was continued by double—stranded DNA
`sequencing (Environmental Genomics Facility. University of
`South Carolina School of Public Health).
`Protein Expryssian and Purification. Recombinant er3
`and er4 were both purified using the previously published
`protocol for er3 (33). We note that recombinant er3 was
`cloned from the second start site (encoding Met36) to the stop
`codon after determining that the first start site (encoding Metl)
`was not utilized in viva (N. Dingra and C. Outten, unpublished
`data). The er3 amino acid sequence numbering in this study
`thus starts with the second start site as Metl. er3 (or er4) was
`coexprcsscd with Fral and Fra2 by transforming pETZla—erS
`(or pETZla-er4) and pRSFDuet-l—Frul-Hiss/FraZ into the
`E. cali strain BLZi (D133). Generally, a l L LB culture was grown
`with shaking at 30 °C and induced with 1 mM iscpropyl
`fi—D—thiogalactoside (IPTG) at ODfim 0.6—0.8. The cells were
`collected by centrifugation 18 h after induction and resuspended
`in 50 mM Tris—MES. pH 8.0,
`followed by sonication
`and centrifugation to remove the cell debris. The cell—free
`extract was loaded onto a DEAE anion-exchange column (GE
`Healthcare) equilibrated with 50 mM Tris-MES. pH 8.0.
`The protein was eluted with a salt gradient. and the fractions
`containing erft (or er4) and Fra2 were pooled and concen-
`trated to 2 mL. A fraction of Fra2 that was not bound to er3
`was also present in the DEAE flow-through and further purified
`as described below. (NH4J2804 was added to the FmZ-er3
`(or Fra2—er4) complex to a final concentration of 1 M, and
`the sample was loaded onto a phenyl-Sepharose column (GE
`Healthcare) equilibrated with 50 mM Tris—MES, pH 8.0,
`100 mM NaCl, and l M (NHs)ZSO4. The protein was then eluted
`with a decreasing (NH4)ZSO4 salt gradient, and the fractions
`containing er3 (or er4) and Fra2 were concentrated and
`loaded onto a HiLoad Superdex 75 gel filtration column (GE
`Healthcare) equilibrated with 50 mM Tris—MES, pH 8.0, and
`150 mM NaCl. The purest fractions ofthe Fm2—er3/4 complex
`as judged by SDS—PAGE were collected and concentrated to
`~250 yL with the addition of 5% glycerol and stored at 780 °C.
`Purification of Fra2-er3/4 was done aerobically: however, the
`procedure was completed in 1 day using degassed buffers to
`minimize loss of the Fe~S cluster.
`STE-labeled samples ofthe Fra2-er3 or Fm2.er4 complex
`for Mbssbauer studies were prepared by growing the E. Cali
`recombinant Fra2-er3 or Fra2-Grief coexpression strain in
`media supplemented with 57FeSO... One liter of cells was first
`grown at 30 °C in LB media to 013m ~06. then collected by
`centrifugation, and resuspended into 1 L of fresh M9 minimal
`media with 0.2% gluconate, The cells were grown at 30 °C for
`30 min; then ”Peso. and WTG were added at 50 pM and 1 mM
`final concentrations. respectively. The cells were collected by
`
`9571
`Biochemistry, Val. 48, No. 40, 2009
`centrifugation 18 11 after induction. Subsequent purification of
`i7Fe-labelcd FmZ-er3 or FraZ-er4 utilized the same protocol
`described in the previous paragraph.
`For purification of Fra2 without er3 or er4, BL21(DE3)
`E. coli cells were transformed with pETZla-Fraz. Fra2 expressed
`in this strain resides in inclusion bodies, thus requiring unfolding
`and subsequent refolding to purify. However, uncomplexed Fra2
`could be purified from the DEAE column flow-through from
`cells coexpressing er3 and F1112 as mentioned above. The Fraz-
`containing DEAE flownhrough fractions were adjusted to pH
`6.0 and loaded onto an SP FF cation-exchange column
`(GE Healthcare) equilibrated with 50 mM MES—Na, pH 6.0.
`Fra2 was eluted with a salt gradient, concentrated, and loaded
`onto a HiLoad Superdex 75 gel
`filtration column (GE
`Healthcare) cquilibrated with 50 mM Tl‘iS'MES. pH 8.0. and
`150 mM NaCl. The purest fractions of Fra2 were collected.
`concentrated to ~500 ”L with the addition of 5% glycerol, and
`stored at ~80 °C. The yield of uncomplexed Fra2 from the
`DEAE flow-through was highest when Fra2 was coexpressed
`with er3(Cl768) (see Results).
`Recombinant her2 was overexpressed in the E. call BL21-
`(DE3) strain and grown at 37 °C with shaking until ODm = 0,6.
`The cultures were cooled to 20 °C, and 1 mM IPTG was added to
`induce her2 expression. After overnight growth (~18 h) at
`room temperature, cells were harvested by centrifugation and
`stored at —80 °C. The cell pellet was subjected to three freeze—
`thaw cycles, and soluble protein was extracted with 50 mM Tris»
`lICI, pH 8.0. The protein was precipitated with 25r60“/u
`(NH4)2S04 and the pellet resuspended in 50 mM Tris-HCI, pH
`8.0, and subsequently loaded on a dcsalting column followed by a
`DEAE column (GE Healthcare) both equilibrated with 50 mM
`Tris-HCL pH 8.0. The majority of herZ did not bind to the
`DEAE column and was concated in the flow-through. These
`fractions were concentrated and loaded onto a HiLoad Superdex
`75 gel filtration column (GE Healthcare) equilibrated with
`50 mM Tris-HCl. pH 8.0, and 150 mM NaCl. [2Fe-ZS] herZ
`elutes as a dimer, while apo herZ elutes as a monomer as
`previously reported (34).
`In Vitro Fe-S Cluster Reconstitution an A110 er3 and
`er4. Reconstitution of an Fe-S cluster on apo er3, 1 mM in
`100 mM Tris-HCl buffer at pH 7.8, was carried under anaerobic
`conditions (02 < 5 ppm) in a glovebox (Vacuum Atmospheres,
`Hawthorne, CA). The reaction mixture involved 2 mM GSl—l, a
`16—fold excess of ferrous ammonium sulfate (”Fe—labeled for
`Mdssbauer samples) and L»cysteine, and catalytic amounts of
`Azotobacter vinelandii NifS (6.27luM) and was incubated at room
`temperature for 50 min. Reagents in excess were removed
`anaerobically by loading onto a High-Trap Q-Scpharosc column
`(GE Healthcare) inside the glovebox. Elution was achieved using
`a NaCl gradient with cluster-bound er3 eluting between 0.60
`and 0.70 M NaCl. Samples were pooled together as a single
`fraction before concentrating and desalting using Ainicon ultra—
`filtration with a YM10 membrane. The same protocol was
`followed for reconstituting an Fe-S cluster on apt) er4.
`Biochemical Analyses. Protein concentrations were deter—
`mined by the Bradford assay [Bio-Rad) using bovine serum
`albumin as the standard. Iron concentrations were determined
`using the colorimetric ferrozine assay (35). Acid—labile sulfur
`concentrations were determined using published methods
`(36, 37). For GSH measurements,
`the purified Fe~S protein
`complexes were denatured and precipitated with 1% 5-sulfosa-
`licylic acid. and GSH in the supernatant was measured by the
`
`
`
`

`

`9572 Biochemistry, Vol. 48, No. 40, 2009
`
`5,5'~dithiobis(2-nitrobenzoic acid)-GSSG reductase cycling
`assay as previously described (38).
`Analytical and Spectroscopic Methods. Analytical gel
`filtration analyses were performed on a Superdex 75 10/300
`GL column (GE Healthcare) equilibrated with 50 mM
`Tris—MES, pH 8.0. and 150 mM NaCl and calibrated with
`the low molecular weight
`gel
`filtration calibration kit
`(GE Healthcare). The buffer was bubbled with N2 overnight
`and degassed to minimize dissolved 02 levels. Elution profiles
`were recorded at 280 nm with a flow rate of 0.5 mL/min.
`UV—visible absorption spectra were recorded using a Beckman
`DU-SOO or Shimadzu [JV—310] spectrophotometer, and CD
`spectra were recorded on identical samples using a lasco J-715
`spectropolarimeter. Mass spectrometry analysis of purified pro—
`teins was determined using a Bruker UltraFlex MALDIaTOF/
`TOF mass spectrometer. A saturated solution ofsinapinic acid in
`50% acetonitrile and 0.1% m‘fluoroacetic acid was used as the
`matrix. and myoglobln and ubiquitin were the calibration
`standards. Resonant» Raman spectra were recorded as pre-
`viously described (39), using an Instruments SA Ramanor
`U1000 spectrometer coupled with a Coherent Sabre argon ion
`laser, with 20yL frozen droplets of l.5~2.6 mM sample mounted
`on the cold finger of an Air Products Displex Model CSA-ZOZE
`closed cycle refrigerator. X-band (~9.6 GHz) EPR spectra were
`recorded using a ESP-300D spectrometer (Bruker. Billeriw,
`MA), equipped with an ESR 900 helium flow cryostat (Oxford
`Instruments, Concord. MA). and quantified under nonsaturating
`conditions by double integration against a 1.0 mM CuEDTA
`standard. Q-band (~35 GHz) CW ENDOR experiments were
`carried out at 2 K using a spectrometer described previously (40).
`Massbauer spectra were recorded by using the previously de-
`scribed instrumentation (41). The zero velocity of the spectra
`refers to the centroid of a room temperature spectrum of a
`metallic Fe foil. Analysis of the Mossbauer data was performed
`with the WMOSS program (SEE Co.).
`X-ray absorption spectra were measured at
`the National
`Synchrotron Light Source (NSLS) beamline X3-B and at Stan-
`ford Synchrotron Radiation Laboratory (SSRL) beamlines 9-3
`and 7-3. Concentrated samples of the FraZ-er3/4 heterodimer
`were mixed with glycerol (final concentration 30% v/v glycerol
`and 1—2 mM Fe) and were frozen in a Lucite sample cell covered
`with Kapton tape. Spectra were collected at cryogenic tempera-
`tures using a helium displex cryostat at NSLS (30 K) or with an
`Oxford Instruments continuous flow liquid helium erysostat at
`SSRL (10 K). Beamlines were equipped with double crystal
`monochromators with Si[l ll] (NSLS) or Si[220] (SSRL) crystals.
`Harmonic rejection mirrors were used. and spectra were collected
`under fully tuned conditions. Canberra solid—state germanium
`detectors (13 element (NSLS) or 30 element (SSRL)) were used to
`detect the iron Ku fluorescence. Data points were collected every
`5 eV in the preedge region. every 0.25 eV in the edge region.
`and every 005 k in the EXAFS region. Scans were typically
`collected for 45 min, with data acquisition time increasing to 5 5
`per point at higher It. Each scan and detector element was
`examined for electronic anomalies and photoreduction effects
`before averaging 6—12 individual scans for analysis. Scans were
`individually calibrated by simultaneously measuring the spec-
`trum of an Fe foil. Scans were energy shifted such that the rising
`inflection of the Fe edge in the foil spectrum was 7111.2 eV.
`Though the Fra2~er3/4 samples were not readily photoreduced,
`we did observe minor shifts in edge energy from scan to scan. As a
`precaution, the beam size was reduced to 1 mm x 3 mm. and new
`
`scans were initiated at fresh locations of the sample surface. At
`this size. six fresh sample spots were monitored per sample.
`Replicate samples were measured with equivalent results.
`Data were processed using the Mac OS 10 version of EX-
`AFSPAK. a suite of data analysis programs available on the
`SSRL Web site. This program was interfaced to Fell‘7.2 to
`generate the theoretical scattering models used in the fits. The
`value of AEO was fixed to a value of ~10 eV and the scale factor
`was fixed at 0.9. values that yield the correct crystallographically
`determined bond lengths and coordination numbers for iron
`model complexes Bond length. R. and the Debye—Waller factor
`(02) were varied freely in each fit. Coordination numbers were
`incremented in fractional steps to refine the optimal number
`based on goodness offit, Fit quality wasjudged using a modified
`F value, F, that adjusts for the number ofvariables used in a fit.
`All fits reported here are to unfiltered data over a k range of l — 14
`A”. Both single scattering and multiple scattering models were
`used to fit the data. In the latter. parameters for an imidazole ring
`scatterer were used to model histidine ligands. For these fits, the
`Fe-N,,,,,d distance and 02 values were floating freely, and the
`other atoms in the imidazole ring were linked to the refined value
`Fe‘Nimid-
`
`RESULTS
`
`Fra2 and er3/4 Copurify as c Heteradimeric Complex.
`To characterize the interactions between Fro2, er3, and er4,
`the individual proteins were initially expressed and purified
`separately for in vitro analysis. Soluble recombinant er3 and
`er4 were easily extracted from E. coli yielding the apo,
`monomeric
`forms upon aerobic purification (Figure
`1,
`Table 1). However, recombinant FraZ was largely found in
`inclusion bodies and partially degraded upon purification
`(Figure I). The molecular mass of the purified proteins (with
`the first Met removed in each case) was continued by MALDI-
`TOF mass spectrometry (Table I). Since Fra2 interacts with Fral
`and er3/4 in vivo (8), we tested whether cocxprcssion of the
`interacting proteins improved the solubility of Fru2. Fral, Fra2,
`and er3 were cocxpressed using an E. coli strain cotrans-
`formed with one plasmid expressing Fral and FraZ and another
`expressing er3. After collecting the induced cells. we immedi-
`ately noted that the cell pellet was a reddish brown color that was
`not observed when the proteins were expressed separately.
`SDS~PAGE analysis showed that Fraz and er3 were both
`expressed at high levels. while the Fral protein was not visibly
`detectable (expected mass = 86.0 kDa with His tag) (Figure 1A).
`Furthermore, Fra2 and er3 were found to copurify as a reddish
`brown-colored complex with a higher apparent molecular mass
`than the individual proteins as determined by analytical gel
`filtration (Figure 13, Table 1). Similar results were obtained
`upon coexpression of Fra2 with er4. The reddish brown Fra2—
`er complex was also purified upon expression of Fra2—er3
`or Fra2-er4 without F