structural evidence for a common zinc binding domain in archaeal and eukaryal transcription factor...

JBIC (2000) 5 : 276±283 � SBIC 2000

ORIGINAL ARTICLE

Christopher M. Colangelo ´ L. Michelle LewisNathaniel J. Cosper ´ Robert A. Scott

Structural evidence for a common zinc binding domain in archaealand eukaryal transcription factor IIB proteins

Received: 11 May 1999 / Accepted: 2 February 2000

Abstract X-ray absorption spectroscopy has beenused to compare the metal coordination of the N-ter-minal zinc binding domain of eukaryal human tran-scription factor (TF) IIB to the previously reportedstructure of archaeal Pyrococcus furiosus (Pf) TFB.Full length and N-terminal fragments for both PfTFBand human TFIIB were cloned, expressed, and puri-fied. The [C10H] variant of PfTFB was constructed toresemble the metal binding motif of higher eukaryalTFIIB proteins by mutating the second cysteine ligandto a histidine. All five proteins bind zinc in a 1 : 1ratio. Zn X-ray absorption spectroscopy of humanTFIIB and [C10H]PfTFB mutant are consistent withZnS3(N,O) ligation, and further suggest that the N/Oligand is an imidazole.

Key words Transcription factor IIB ´ Metalloprotein ´Zinc finger ´ Gene transcription ´ X-ray absorptionspectroscopy

Abbreviations BRE: TF(II)B recognition element ´ESI-MS: electrospray ionization-mass spectrometry ´E. coli: Escherichia coli ´ EXAFS: extended X-rayabsorption fine structure ´ hTFIIB: human transcrip-tion factor IIB ´ hTFIIBn: N-terminal 59 residues ofhuman transcription factor IIB ´ ICP-AES: inductivelycoupled plasma-atomic emission spectroscopy ´LB: Luria-Bertani media ´ P. furiosus: Pyrococcusfuriosus ´ PfTFB: P. furiosus transcription factor B ´PfTFBn: N-terminal 50 residues of P. furiosus tran-scription factor B ´ RNAP: RNA polymerase ´ SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel

electrophoresis ´ TBP: TATA binding protein ´TFB: transcription factor B ´ XAS: X-ray absorptionspectroscopy ´ XANES: X-ray absorption near edgestructure

Introduction

To date, five archaeal genomes have been fully char-acterized and eight more are currently listed as inprogress [The Institute of Genomic Research (TIGR)Microbial Database, http://www.tigr.org/tdb/mdb/mdb.html]. These results have led to a greater under-standing of the archaeal phylogenetic domain and itsrelationship to both bacterial and eukaryal domains[1, 2]. Initially, ribosomal RNA comparisons showedthat Archaea were more closely related to the Euka-rya and placed on a separate branch from Bacteria inthe universal phylogenetic tree [3±5]. This is mostclearly documented in their transcription systems.Although all three domains have homologous RNApolymerase systems, a much higher similarity existsbetween the archaeal and eukaryal versions thanbetween either of these and the bacterial version.

Transcription factor (II)B [TF(II)B] is one of theconserved transcription components between Archaeaand Eukarya [6, 7] and is required for transcription ini-tiation at protein coding genes [8, 9, 10, 11]. EukaryalTFIIBs have been identified in human [12], Rattus nor-vegicus [13], Xenopus [14], Drosophila [15], Arabidop-sis [16], Glycine max [16], Caenorhabditis elegans [17],Saccharomyces cerevisiae (baker's yeast) [18], andKluyveromyces lactis (yeast) [19], while archaeal TFBshave been found in Haloferax volcanii (GenbankAccession AAD43074), Pyrococcus furiosus [20], P,woesei [6], P. abyssii (Genbank Accession CAB49598),P. horikoshii [21], Aeropyrum pernix [22], Archaeoglo-bus fulgidus [23], Methanobacterium thermoautotrophi-cum [24], Mc. janaschii [25], and Sulfolobus shibatae[26]. TF(II)B contains two functional units, the N- andC-terminal domains, that interact with promoter DNA,

C.M. Colangelo ´ L.M. Lewis ´ N.J. Cosper ´ R.A. Scott ())Department of Chemistry and Center for MetalloenzymeStudies, University of GeorgiaAthens, Georgia 30602-2556, USATel: +1-706-5422726Fax: +1-706-5429454e-mail: [email protected]

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other general transcription factors, and RNA polymer-ase II [10, 12]. The C-terminal domain (CTD) struc-ture in both P. woesei TFB (PwTFBc) and humanTFIIB (hTFIIBc) (Fig. 1c) have nearly identical folds,resembling two cyclin A-like domains. The twodomains have positively charged surfaces that makedirect contact with both TATA binding protein (TBP)and DNA residues upstream and downstream of theBoxA/TATA-box sequence [27±29]. Also, the helix-turn-helix domain in the second repeat of the C-termi-nus is essential for both binding the TFIIB recognitionelement (BRE) and orienting the transcription com-plex on promoter DNA [30]. The N-terminal domaincontains a zinc finger-like motif that is required forassociation of RNA polymerase II/transcription factorIIF (RNAPII/TFIIF) with the transcription complex[31, 32, 33]. NMR studies of P. furiosus TFB N-ter-minal domain (PfTFBn) [34] have shown that theN-terminal domain folds into a ªzinc ribbonº structure(Fig. 1b) with striking similarity to the C-terminaldomain of eukaryal transcription elongation factorTFIIS [35, 36]. Previously, X-ray absorption spectros-copy (XAS) was used to characterize the Zn (or Fe)binding site, a CysX2CysX15 ± 17CysX2Cys motif, of thezinc ribbon in PfTFBn [20].

In this report, XAS is used to characterize theN-terminal zinc-binding site of a eukaryal (human)TFIIB, and the results are compared with our previousanalysis of the archaeal PfTFB Zn [20]. These motifsdiffer in that the second conserved cysteine of theCysX2CysX15±17CysX2Cys motif is replaced by histid-ine in higher eukaryal homologs (Fig. 1a). We showby XAS that this substitution yields the expected liga-tion change to ZnS3(N,O)1 coordination. Engineeringa CysX2HisX15CysX2Cys motif into the archaeal TFB

N-terminal domain (in [C10H]PfTFB) results in thesame zinc coordination change.

Materials and methods

Taq DNA polymerase and deoxyribonucleotides were purchasedthrough Perkin-Elmer (Foster City, Calif.). Primers wereobtained from Integrated DNA Technology (Coralville, Iowa)and Escherichia coli (E. coli) strains 71-18 and BL21(DE3) wereobtained through Novagen (Milwaukee, Wis.) and New EnglandBiolabs (Beverly, Mass.), respectively. Restriction enzymes andT4 DNA polymerase were obtained from either New EnglandBiolabs, Promega (Madison, Wis.), or Boehringer Mannheim(Indianapolis, Ind.). Phenol/chloroform and agarose were pur-chased through Amresco (Solon, Ohio), agar and Luria-Bertani(LB) medium from Difco Laboratories (Detroit, Miss.), andMini-Prep and Midi-Prep plasmid purification kits from Qiagen(Valencia, Calif.). All other chemicals were purchased throughSigma (St. Louis, Mo.) or Fisher (Pittsburgh, Pa.).

PfTFB, PfTFBn, and [C10H]PfTFBn

Cloning, expression, purification, and characterization of PfTFBand PfTFBn were carried out as previously described [20, 34].[C10H]PfTFBn was cloned using site-directed polymerase chainreaction (PCR) mutagenesis with PfTFB/pT7-7 as the template.After ligation of the restricted PCR product with pT7-7 [37], theplasmid was transformed into E. coli BL21(DE3) for inducibleexpression. The soluble protein was released from the cells bysonication in 20 mM Tris, pH 8.0, and the clarified supernatantloaded onto a Q-sepharose column (Pharmacia). The proteineluted with 250 mM NaCl and was subsequently applied to aMonoQ 10/10 column (Pharmacia) for purification to homoge-neity.

Transformation and expression of human TFIIB gene

We obtained the phIIB plasmid, containing the gene for humantranscription factor IIB (hTFIIB), as a generous gift from D.

Fig. 1 a Schematic sequencealignment for human TFIIB,human TFIIBn,[C10H]PfTFBn, PfTFBn, andPfTFB. b Ribbon drawing ofPfTFB N-terminal domainNMR structure [34]. c Ribbondrawing of human TFB C-ter-minal domain NMR structure[29]

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Reinberg of the Howard Hughes Medical Institute, Rutgers, NJ[12]. The plasmid was transformed into E. coli strain JM109,and colonies were purified using Midi-Prep (Qiagen) andsequenced at the Molecular Genetics Instrumentation Facility(MGIF), University of Georgia. For expression, the phIIB plas-mid was transformed into E. coli strain BL21(DE3), which wasgrown at 37 �C on LB medium supplemented with ampicillin(0.1 g/L), until the optical density at 600 nm reached 0.6, atwhich time both isopropylthio-b-D-galactoside (IPTG) (0.1 g/L)and zinc chloride (50 mg/L) were added. The temperature wasreduced to 30 �C, and the cells were allowed to grow for 5 hbefore harvesting.

Construction and expression of the gene for human TFIIBN-terminal domain (hTFIIBn)

Using plasmid phIIB, primers were designed for PCR amplifica-tion of the first 59 residues of hTFIIB. The primers containedeither an NdeI [hTFIIBNTDForward (59-ATA TAC AT-A TGG CTT CTA CCA GC-39)] or a BamHI [hTFIIBNTD-Reverse (59-TAC CGG ATC CTT ATT TGT CAT TGC T-39)]restriction site for cloning into the pT7-7 vector. ªHot startºPCR amplification was performed according to the manufac-turer's instructions for 60 cycles of 94 �C for 2 min, 60 �C for2 min, and 72 �C for 4 min. After PCR amplification, the197-bp product was inserted into the pT7-7 plasmid [37] usingrestriction overlap cloning [38]. Briefly, both PCR product andplasmid were double digested with NdeI and BamHI, bufferexchanged via ethanol precipitation extraction, and ligated withT4 DNA ligase. Positive inserts were selected using SmaI andthen transformed into E. coli 71-18. Positive colonies were iden-tified using alkaline lysis Mini-prep [38] and restriction enzymesites within both the gene and plasmid (HindIII and BglII,respectively). Plasmids from positive colonies were purifiedusing Midi-Prep and sequenced at the MGIF. Expression in E.coli BL21(DE3) was carried out as described above.

Purification of hTFIIB

After harvesting cells from a 2 L growth, the hTFIIB cell pelletwas stored at ± 20 �C. The cells were thawed at room temper-ature, and resuspended in 70 mL of buffer A (20 mM Tris,pH 7.9, 5 mM DTT, 100 mM KCl, and 20% glycerol) with70 mL each of 0.6 mM leupeptin, 100 mM PMSF, and 70 mMlysozyme. After incubation at room temperature for 10±20 min,the solution was sonicated four times on ice (Fisher Scientific550 Sonic Dismembrator) for 30 s (1 s on, 1 s off) at 1 minintervals. The supernatant was clarified by spinning at 15,000gfor 30 min three times. The supernatant was loaded onto anS-Sepharose column equilibrated with buffer A and then elutedwith 60% buffer B (buffer A+1 M KCl). The eluent was concen-trated using an Amicon (Beverly, Mass.) YM30 membrane.

Purification of hTFIIBn

Harvested cells from a 2 L growth were resuspended in 45 mLof buffer C (10 mM Tris buffer, pH 7.5) and 180 mL of EDTA(0.5 M) was added, yielding a final concentration of 2 mM.Then 70 mL of both 100 mM PMSF and 0.6 mM leupeptin wereadded as protease inhibitors; 45 mL RNAse (0.5 mg/mL) and22.5 mL DNAse (0.5 mg/mL) were also added to degrade resid-ual DNA and RNA. Cells were sonicated and pelleted asdescribed above. After centrifugation, clarified supernatant wasloaded onto a QAE Sephadex A-25 column (Pharmacia) equili-brated with buffer C, washed with buffer C+50 mM NaCl, andeluted with buffer C+400 mM NaCl. The eluent was concen-trated and de-salted using an Amicon YM3 membrane, thenloaded onto a 10/10 Mono Q (Pharmacia) column. Two peakseluted between 20% and 30% with buffer C+1 M NaCl.

Protein characterization

Protein concentrations of hTFIIB, hTFIIBn, and [C10H]PfTFBnwere estimated by absorbance at 280 nm using an extinctioncoefficient calculated on the basis of translated amino acid com-position [39]. UV-visible spectra were recorded on a Shimadzu(Columbia, Md.) UV2101PC scanning spectrophotometer. Pro-tein molecular masses were determined by electrospray ioniza-tion-mass spectrometry (ESI-MS) on a PE Sciex API 1 Plusmass spectrometer (Foster City, Calif.) at the Chemical and Bio-logical Sciences Mass Spectrometry Facility, University of Geor-gia. Metal concentrations were determined using inductivelycoupled plasma-atomic emission spectroscopy (ICP-AES) at theChemical Analysis Laboratory, University of Georgia.

X-ray absorption spectroscopy

For XAS, about 160±180 mL of each sample was transferred to apolycarbonate cuvet (24 � 4 � 2 mm) covered with 0.001 inchthick Mylar adhesive tape as an X-ray transparent window mate-rial. The cuvet was capped and dropped into liquid nitrogen. ZnK-edge XAS data were collected on beamline 7-3 at the SSRLwith the SPEAR storage ring operating at 3.0 GeV and60±100 mA current [40] (Table 1). Standard extended X-rayabsorption fine structure (EXAFS) analysis was performed [40]using EXAFSPAK software (http://ssrl01.slac.stanford.edu/exaf-spak.html). When necessary, both single- and multiple-scatteringpaths up to 4.5 � from the Zn atom were used to identify andquantify imidazole coordination due to histidine. Multiple-scat-tering paths were built from a model taken from the crystallo-graphic coordinates of tetrakis(imidazole)zinc(II) perchlorate[41]. The coordinates were imported into Ball & Stick (v. 3.5,Cherwell Scientific) and edited to only the metal atom and oneimidazole. The coordinates were then imported into FEFF v.7.02 software [42] to calculate scattering amplitudes and phaseshifts for each scattering path containing four or fewer legs. Aconstrained fitting process was then used with the following

Table 1 XAS data collection and reduction

SR facility SSRL

Beamline 7-3

Monochromator crystal Si [220]

Detection method fluorescence

Detector type solid-state arraya

Scan length (min) 23

Scans in average 12

Temperature (K) 10

Energy standard Zn foil (first inflection)

Energy calibration (eV) 9659

E0 (eV) 9670

Pre-edge backgroundEnergy range, eV 9333±9625Fit type GaussianGaussian center (eV) 8720Width (eV) 1000

Spline backgroundEnergy range (eV) 9670±9881(4)(Polynomial order) 9905±10093(4)

10093±10305(3)

a The 13-element Ge solid-state X-ray fluorescence detector atSSRL is provided by the NIH Biotechnology Research Resource

279

parameters. Coordination numbers were constrained to beinteger values. The distances (Ras) and Debye-Waller factors(sas

2 ) for outer-shell atoms of imidazole rings were constrainedto a given ratio with first-shell (metal-nitrogen/oxygen) distanceand the Debye-Waller factor, respectively. First-shell distancesare expected to be accurate to within � 0.02 �. The bondvalence sum method was applied to EXAFS fits using standardprocedures [43±47].

Results and discussion

Previously, we showed that archaeal PfTFB binds zincin a CysX2CysX15CysX2Cys zinc-ribbon structure [20,34]. This led to our general working hypothesis thatthe CysX2HisX17CysX2Cys motif in the N-terminaldomain of the higher eukaryal (e.g., human) TFIIBproteins binds zinc in the same overall fold as theCysX2CysX15CysX2Cys zinc-ribbon structure of theN-terminal domain of the archaeal (e.g., P. furiosus)TFB proteins [20]. To test this hypothesis, twoTF(II)B proteins were cloned, hTFIIB and PfTFB(Fig. 1a). Full-length human (h)TFIIB exhibits 32%identity (56% similarity) with full-length PfTFB, a34% identity (55% similarity) of their N-terminaldomains, and a 31% identity (47% similarity) of theirC-terminal domain (CTD). For structural studies, wesubcloned the N-terminal domains of these two pro-teins (Fig. 1a), and also engineered the higher euka-ryal CysX2HisX15CysX2Cys motif into the archaealsequence by constructing [C10H]PfTFBn.

Protein characterization

The five proteins, cloned, expressed, and purified asdescribed in Materials and methods, were determinedto be at least 95% pure and monomeric by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and gel filtration, respectively. Owing toprotein solubility problems with the two full-lengthproteins, only the N-terminal fragments were furthercharacterized via mass spectrometry and metal analy-sis. The ESI-MS of hTFIIBn revealed one series ofmolecular ions, deconvoluted to a mass of 6361 Da,which corresponded to hTFIIBn without an N-terminalmethionine (calculated as 6362.06 Da). The ESI-MSof [C10H]PfTFBn revealed two series of molecularions with deconvoluted masses of 5668 and 5537 Da,which corresponded to [C10H]PfTFBn with and with-out an N-terminal methionine (calculated as 5669.43and 5538.23 Da), respectively. Metal/protein ratioswere determined using UV-vis spectroscopy to meas-ure the protein concentration (280 nm) and ICP-AESto measure the zinc concentration. Zn/protein ratiosof 0.996 � 0.005 for hTFIIBn and 0.885 � 0.009 for[C10H]PfTFBn were calculated, which confirm thatboth proteins bind metal in a 1 : 1 ratio. These resultsare comparable with previous metal/protein deter-minations for PfTFBn [20].

X-ray absorption near edge structure (XANES)

The Zn K-edge spectra of all three N-terminal proteindomains show distinct maxima at ~ 9663 and~ 9670 eV (Fig. 2), but differences between theXANES spectra of PfTFBn and both [C10H]PfTFBnand hTFIIBn are seen in the relative intensity of thesepeaks. Comparison with previous reports on bothmodel compounds and peptide complexes suggeststhat the relative increase in the intensity of the~ 9670 eV maximum accompanies substitution of sul-fur by nitrogen ligands [48, 49]. The XAS edgechanges we observe are consistent with PfTFBn con-taining four sulfur ligands and both [C10H]PfTFBn orhTFIIBn having three sulfur and one nitrogen ligands.

Extended X-ray absorption fine structure

The Zn K-edge EXAFS of hTFIIBn and[C10H]PfTFBn are similar in phase and frequency tothe EXAFS of PfTFBn, but exhibit significantly loweramplitude than PfTFBn (Fig. 3a). This difference isvisualized as a decrease in the ca. 2.3 � Fourier trans-form (FT) peak (Fig. 3b). A decrease would beexpected if the histidine in [C10H]PfTFBn and hTFI-IBn has replaced one of the four cysteine ligands inPfTFBn. Metal-ligand distances correlate with metaloxidation state (valence), coordination number, andligand type. The bond valence sum (BVS) value is cal-culated from metal-ligand distances and coordinationnumbers from a given EXAFS fit and shouldapproach the metal valence (+2 for Zn) if the fitparameters make sense. BVS values calculated forthree-coordinate Zn fts (fit 5, Table 2, for example)are too low, compared to values for all four-coordi-nate fits, suggesting that all zinc sites are four-coordi-nate (Table 2). Only slight improvements in the good-

Fig. 2 X-ray absorption edge spectrum for Zn forms of PfTFBn(±±±) [20], [C10H]PfTFBn (- - -) and human TFIIBn (......).Human TFIIB data (not shown) are essentially identical to thoseof human TFIIBn

280

ness-of-fit values (f9) result if ZnS3(N,O)1 simulationsare compared to ZnS4 simulations. These improve-ments are 4±5% for PfTFB and PfTFBn (fits 2 vs. 1, 4vs. 3, Table 2) but 16±21% for the other (fits 7 vs. 6,10 vs. 9, 13 vs. 12, Table 2). However, ZnS4 fits forPfTFBn (fit 3, Table 2) yield similar sas

2 values asZnS3(N,O)1 fits for hTFIIBn or [C10H]PfTFBn (fits 7,

11, Table 2), suggesting that three of the four ligandsto Zn in the latter two proteins are sulfur. Histidineimidazole coordination is usually reflected in outer-shell FT peaks at 3 and 4 �. Such peaks are notobviously present in the Zn EXAFS FTs for hTFIIBnor [C10H]PfTFBn, compared with that for PfTFBn(Fig. 3b). Nevertheless, we attempted curve-fitting of

Table 2 Curve-fitting resultsfor Zn EXAFSa Sample Fit Group Shell Ns Ras sas

2 DE0 f9b BVSg

(�) (AÊ 2) (eV)

PfTFB 1 S Zn-S (4)d 2.33 0.0030 ± 4.82 0.075 2.09< 0.041>c

2 S Zn-S (3) 2.33 0.0009f ± 5.31 0.071 2.07N/O Zn-N (1) 2.03 0.0022 [ ± 5.31]e < 0.029 >

PfTFBn 3 S Zn-S (4) 2.33 0.0020 ± 5.09 0.056 2.09< 0.034 >

4 S Zn-S (3) 2.34 0.0004 ± 3.60 0.054 1.93N/O Zn-N (1) 2.11 0.0016 [ ± 3.60] < 0.030>

[C10H]PfTFBn 5 S Zn-S (3) 2.32 0.0026 ± 4.54 0.066 1.61< 0.056>

6 S Zn-S (4) 2.32 0.0045 ± 4.88 0.067 2.15< 0.060>

7 S Zn-S (3) 2.32 0.0022 ± 5.62 0.053 2.14N/O Zn-N (1) 2.01 0.0023 [-5.62] < 0.042>

8 S Zn-S (3) 2.32 0.0022 ± 4.89 0.042 2.14imid Zn-N (1) 2.01 0.0019 [ ± 4.89] < 0.032 >imid Zn-C2 (1) [3.00] [0.0028] [ ± 4.89]imid Zn-C3 (1) [3.07] [0.0029] [ ± 4.89]imid Zn-N4 (1) [4.16] [0.0039] [ ± 4.89]imid Zn-C5 (1) [4.20] [0.0040] [ ± 4.89]

Human TFIIBn 9 S Zn-S (4) 2.32 0.0039 ± 5.31 0.057 2.15< 0.052 >

10 S Zn-S (3) 2.33 0.0018 ± 5.42 0.048 2.07N/O Zn-N (1) 2.03 0.0033 [ ± 5.42] < 0.038 >


Human TFIIB 12 S Zn-S (4) 2.32 0.0039 ± 5.12 0.060 2.15< 0.050 >

13 S Zn-S (3) 2.32 0.0017 ± 5.20 0.050 2.11N/O Zn-O (1) 2.03 0.0026 [ ± 5.20] < 0.038 >


a Group is the chemical unit defined for the multiple scattering calculation. Ns is the number ofscatterers (or groups) per metal. Ras is the metal-scatterer distance. sas

2 is a mean square deviationin Ras. DE0 is the shift in E0 for the theoretical scattering functions. A k-range of 2.0 ± 12.0 � ± 1

was used for all data sets.b f9 is a normalized error (chi-squared):

f0 �P

ik3 wobs

i ÿ wcalci

ÿ �� 2=N

� �1=2

k3wobs� �maxÿ k3wobs� �min

� � �2�

c Numbers in angle brackets are f' for smoothed datad Numbers in parentheses were not varied during optimizatione Numbers in square brackets were constrained to be a multiple of the value abovef Underlined numbers contain values that are chemically or physically unreasonableg BVS = Ss, s = exp[(r0 ± r)/B], B = 0.37, r0(Zn2+-N) = 1.776, r0(Zn2+-S) = 2.09

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the Zn EXAFS data using ZnS3(imidazole)1 coordina-tion and found some improvement in f9 and reasona-ble sas

2 values for the outer-shell imidazole scatterers(fits 8, 11, 14, Table 2). The curve-fitting resultsclearly show that all TF(II)B protein Zn sites are fourcoordinate. Human TFIIB, human TFIIBn, and[C10H]PfTFBn are best fit with either a ZnS3(N,O)1 orZnS3(imid)1, and PfTFBn and PfTFB are best fit byZnS4 coordination.

Ligand distribution analysis

One of the common limitations in EXAFS is theinability to quantify the presence of light (low-atomicnumber) backscatterers (e.g., N, O) in the presence ofheavier backscatterers (e.g., S). Sulfur backscatteringalways dominates the EXAFS and in most casesobscures the nitrogen/oxygen backscattering. This isespecially problematic in determining whether anyN/O-containing ligands exist in a coordination spheredominated by S-containing ligands, since someimprovement in the two-shell MS3(N,O)1 fit must beattributed to the additional degrees of freedom con-tributed by the second-shell parameters. When work-ing with dilute biological samples, the interpretableEXAFS k-range is usually limited to ~ 12±13 � ± 1.Limited resolution makes it difficult to discriminateseparate N and S shells.

Penner-Hahn and co-workers [49] have developeda comparative curve-fitting analysis that attempts tocorrect for improvements in two-shell fits due to the

increase in variables. Mixed coordination fitsMSx(N,O)4 ± x are compared with split-shell MS2S92 fitswhich artificially measure improvement from just theincrease in the number of parameters. In our applica-tion of this procedure, we obtained raw XAS data ofthe Zn inorganic compounds used in the originaldevelopment from Penner-Hahn and co-workers [49].The raw data sets were re-processed as described inthe Materials and methods section to ensure internalconsistency and the EXAFS were fit by allowing Rasand sas to vary for each shell while holding all otherparameters fixed. For each data set, a series ofZnSxN4 ± x fits were performed in which x was variedin increments of 0.1 (x=0.8±4.0). For each fit, the per-cent improvement relative to the two-shell 2S+2S9 fit,Pi, was calculated using Eq. 1:

Pi � f �MS2S20� ÿ f MSxN4ÿx� �f MS4� � �1�

where f is the uncorrected root-mean-square fit index,the numerator of the expression in footnote b,Table 2. Figure 4 displays Pi as a function of x (con-verted to percent sulfur) for the Zn model compoundsand the three N-terminal domains of TF(II)B proteins.A maximum in the Pi versus %S curve should indicatethe best-fit distribution of sulfur and nitrogen/oxygenligands.

For the zinc compounds, the Pi versus %S curvesexhibit maxima at about 50% S for ZnS2N2, and about75% for ZnS3N. There is no maximum for ZnS4, inaccord with previously published results [49]. Thebehavior of these plots for both hTFIIBn and

Fig. 3 k3-weighted EXAFS (top) and phase-corrected Fouriertransforms (FT, bottom) for Zn forms of PfTFBn (±±±) [20],[C10H]PfTFBn (- - -) and human TFIIBn (......). Human TFIIBdata (not shown) are essentially identical to those of humanTFIIBn

Fig. 4 Percent improvement, Pi, plotted as a function of percentsulfur in the Zn EXAFS fits. Dotted lines represent third-orderpolynomial fits to the model complexes for ZnS4 (open circles),ZnS3N (open triangles), and ZnS2N2 (open squares). Solid linesrepresent third-order polynomial fits for PfTFBn (solid trian-gles), hTFIIBn (solid squares), and [C10H]PfTFBn (solid circles).Human TFIIB data (not shown) are essentially identical to thoseof human TFIIBn. The negative Pi values for the ZnS4 modelcomplex reflect the fact that all of the S+N fits are worse thantwo-shell 2S+2S fits

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[C10H]PfTFBn most resembles that of the ZnS3Nmodel, showing a maximum at about 75% sulfur,while the plot for PfTFBn resembles that of the ZnS4model. This analysis provides support for our assign-ment of ZnS3N coordination for hTFIIBn and[C10H]PfTFBn.

Conclusion

XAS analysis of human TFIIB indicates that theN-terminal domain, which is conserved among archaeaand eukarya, binds zinc in a ZnS3(N,O)1 coordinationenvironment. A similar Zn binding site can be engi-neered into P. furiosus TFB by introducing the[C10H] mutation, and the resulting Zn site structure isessentially identical to that in human TFIIB. Theseresults suggest that the higher eukaryal TFIIB proteinshave a similar fold in their N-terminal domains as thezinc ribbon of PfTFBn [20], and that this structuralfeature is conserved throughout evolution. NMR stud-ies are in progress to elucidate the overall protein foldin hTFIIBn.

Acknowledgements J. E. Penner-Hahn graciously provided Zncompound XAS data for comparison in Fig. 4. This work is sup-ported by the National Science Foundation (MCB-9631093).This work was (partially) supported by the NSF Research Train-ing Group Award to the Center of Metalloenzyme Studies (DIR90-14281); C.M.C., L.M.L., and N.J.C. received CMS RTGTraineeships. The XAS work is supported by the National Insti-tutes of Health (GM 42025). The XAS data were collected atthe Stanford Synchrotron Radiation Laboratory (SSRL), whichis operated by the Department of Energy, Division of ChemicalSciences. The SSRL Biotechnology Program is supported by theNIH, Biomedical Resource Technology Program, Division ofResearch Resources. Support for the X-ray fluorescence detec-tor is from NIH BRS Shared Instrumentation Grant RR05648.

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structural evidence for a common zinc binding domain in archaeal and eukaryal transcription factor...

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