j. biol. chem.-1992-patrick-6910-5

7/29/2019 J. Biol. Chem.-1992-Patrick-6910-5

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THE OURNAL F BIOLOGICALHEMISTRY0 1992by TheAmericanSocietyfor Biochemistry andMolecular Biology, nc. Vol. 267, No. 10, lseueof April 5,pp.6910-6915,1992Printed inU. .A.

Characterizationof Functional HPV-16E7Protein Produced inEscherichiacoli(Received for publication, October 18, 1991)

DenisR. Patrick, Ke ZhangS, DeborahDefeo-J ones, GeraldR . Vuocolo, RobertZ. Maigetters,Mohinder K . Sardanaq, Allen Oliff1 and David C. HeimbrookFrom theDepartments of Cancer Research, Cellular and M olecularBwlogy, and lBiologica1 Chemistry, Merck Sharp andDohme Research Laboratories, West Point, P ennsylvania19486and the$Bwstructures Institute, U niversity Ci ty Science Center,Philadelphia, Pennsylvania 19104

Human papillomaviruses HPVs)are he etiologicagents responsible for genital warts and are contrib-uting factors n the pathogenesis of human cervicalcancer. TheHPV E7gene is transcriptionally active inthese diseasesand has been shown to transformam-malian cells in vitro.W e have expressed and purifiedthe HPV-16E7gene product in Escherichia coli. TheisolatedE7protein contains zinc in a :l molar ratio.X-ray absorption fine structure studies demonstratedthat the zinc is coordinated by 4 sulfur igands. Wesequentially derivatized theE7 cysteines to differen-tiate between solvent-exposed, metal-bound, and di-sulfide-associated cysteines. Our results demonstratethat CysZ4 and Cyses are accessible to solvent, whilecysteines in the wo conserved Cys-X-X-Cys motifsrelikely involved in binding zinc. We observed no evi-dence for the existence of disulfide bonds in recombi-nantE7protein under the conditions tested.

Nearly half of the known human papilloma viruses (HPVs)can infect the genital mucosa and produce benign epitheliallesions. Epidemiological and molecular genetic studies havedemonstrated that only a few of the HPV solates that infectthe genital mucosa are associated with most (75-100%) casesof cervical cancer (1). In these cases two viral genes, E6 andE7, are transcriptionally active (2). The most common HPVstrain associated with cervical neoplasia is HPV-16. TheHPV-16 E6 and E7 genes together have been shown to benecessary and sufficient for transformation of primary humankeratinocytes (3). The HPV-16 E7 gene is able to transformestablished rodent fibroblasts (4), and, n conjunction withras, primary rat epithelial cells (5).The HPV-16 E7 gene encodes a protein of 98 amino acids(6) which shares a region of homology (amino acids 2-35)with the adenovirus E1A protein and simian virus-40 largeTantigen. E7, like ElA, can transactivate transcription from

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*The costs of publication of this article were defrayed in part bythe payment of page charges. T his article must therefore be herebymarked advertisement in accordance with 18 U.S.C . Section 1734solely to indicate this fact.(1 T owhom correspondence should be addressed Bldg. WP16-101,M SDR L , W est Point, PA 19486. Tel.: 215-661-3074; Fax: 215-661-7320.T he abbreviations used are: H PV , human papillomavirus;EXAFS, extended x-ray absorption fine structure; DT T , dithiothre-itol; R DF, radial distribution function; IAM, iodoacetamide; 4-VP, 4-vinylpyridine; HPL C, high performance liquid chromatography;HEPES, 4- ( 2- hydroxyethyl ) - 1- pi perazi neet hanesul f oni ccid; pRB,retinoblastoma growth suppressor ene product; SDS, sodium dodecylsulfate.

the adenovirus E2 promoter (7, 8).The E7 protein has alsobeen shown to associate with the retinoblastoma growth sup-pressor gene product pRB (9,lO). The shortegion of limitedhomology between E7, ElA,and large T is required forefficient transformation (11-13) and pRB inding (10,12,14-16). Five of the 7 cysteine residues of HPV E7 proteins arehighly conserved and have been the targets of numerousmutagenesis studies (16-18). Single amino acid substitutionsof cysteine with glycine at positions 24,58,61,91, or 94greatly reduced or completely abolished the transformingactivity of HPV E7. The cysteine at position 24 is in thecenter of the conserved pRB binding domain, E7(20-30) (19).The other 4 conserved cysteines in E7 are contained in Cys-X-X-Cys repeats which have been suggested to mediate zincbinding, although they lack some of the conserved aminoacids and size features of the zinc finger DNA bindingsequences (20). Barbosa et al. (21) have shown that the E6and E7 proteins of HPV-18 bind zinc when immobilized onfilters. Rawlset aZ. (7), using the same technique, have shownthat chemically synthesized HPV-16 E7 can bind zinc, and aprotein containing only one of the Cys-X-X-Cys motifs wasstill able to bind zinc and trans-activate gene expression fromthe E2 promoter. These results suggest that residues otherthan the ysteines may be involved in metal ion coordination,and that the onserved cysteines might have functions otherthan zinc binding.We report here the purification and characterization ofbacterially expressed HPV-16 E7 protein. The purified pro-tein is capable of binding to the retinoblastoma growth sup-pressor protein. The E7protein binds1mol of zinc ion/molof protein via four sulfur ligands. A sequential derivatizationscheme was developed o determine the physical properties ofeach cysteine residue of the protein in solution. We found noevidence of disulfide bridge formation. Our results are con-sistent with the hypothesis that residues Cys5, Cys61, Cys,and Cysg4bind zinc in thenative protein, while residue CYS*~is available for interaction with pRB.

MATERIALS AND METHODSEscherichia coli Expression and Purification of HPV-16 E7 Pro-tein-The H PV -16 E7 gene (Peter Howley, National Institutes ofHealth) was cloned into the pTACSDvector described by L inemeyeret al. (22). T he 16-amino acid extension was cloned in from thepUC18 vector (F ig. 1). Constructs lacking the N-terminal extensionsequence had greatly r educed levels of expression. Expression plas-mids were transformed nto DH5aIqE. coli cells (BRL ).E. coli cell paste expressing HP V-16 E 7 was generated by dilutinga mid-logarithmic overnight culture of E. coli containing theTACSDE 7202 lasmid 1:lOO into a 10-liter fermentor containing 3 XL S media (15 g/li ter yeast extract, 30 g/liter soya peptone, 10 g/literNaC l, and 100 mg/liter ampicill in). T he cells were grown at 37 C,90liter/min air-flow, 260 rpm for 6 h; when they reached mid-log (Am

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Characterization of HPV-16 E7 Protein 6911=5), i sopropyl - 1- t hi o- P-D- gal act opyranosi deas added to 1 mM. Thecells were harvested 2 h post-induction,yielding approximately 130gof cell paste.In a typical purification run, frozen cell pellets (62 g total) wereresuspended in 400 m of 50 mM Tri s, pH=7.5, 50 mM NaCI, 5mMDTT, and 3 mM zinc acetate. Lysiswas achieved by three 2-minbursts of sonication on ice with stirring in hetween bursts (Fig. 1R,lane 1 ) . Urea was added to a final concentration of 8 M to solubilizethe E7 protein (about 50%of the E7protein was insoluble withoutthe urea). Insoluble material was emoved by centrifugation. Thesupernatant (Fig. lR, lane 2) was loaded onto a 5 X 23-cm DEAE-Sepharose column equilibrated inM urea, 50 mM Tris,50mM NaCI,1mM DTT, and0.1 mM zinc acetate, pH=7.5. Bound proteins wereeluted with a 3-l iter 50-400 mM NaCl gradient, and fractions con-taining E7 were pooled (see Fig. lR, lane 3). The E7 protein wasrefolded by diluting thepooled sample 1:4 with water, adding educedand oxidized glutathione to a final concentration of 1and 0.25 mM,respectively, and adjusting thepH to 9.0. After overnight incubationat 4 "C, the refolded protein was concentrated on an Amicon stirredcell concentrator to avolume of 150 ml and dialyzed against 50mMTris, 50mM NaCI, 1mM DTT, and 0.1 mM zinc acetate, pH =7.6.Aliquots of the refolded sample (25 ml)were loaded onto a 2.5X 100-cm (3-100 column equilibrated in the sameuffer at 4 "C. Late-elutingfractions containing E7 were pooled (Fig. lR, lane 4 ) , yielding ap-proximately 60 mg of E7 protein. Gel filtration standards ndicatethe refolded E7 tohebetween 40 and 60kDa in size.ExtendedX-ray Absorption FineStructure (EXAFS) Experi-ments-EXAFS sastructural echnique which probes he localenvironment of metal ons (23). Basically, x-ray absorption occurswhen the x-ray energy is just sufficient to promote an electron froman atomic bound state to a propagating continuum state in the metalion. The scattering of the photoelectron by the atoms surroundingthe metal ion induce an interference pattern,which modulates the x-ray absorption spectrum. By mathematical deconvolution and com-parison with model compounds, the oscillations in the ahsorptionspectrum can provideprecise structural nformation on the metalligands, such as their identity, number, and interatomic distance.EXAFSexperiments were conducted at the Riostructures PRTBeamline X9-A of the National Synchrotron L ight Sourceat Brook-haven National Lab. A Si(220) double crystal monochromator and aharmonic rejection mirror were used for theexperiments.A 13-element germanium detector was used to collect zinc K-edge fluores-cence and to reject x-ray photons at otherenergies. The HPV -16 E7protein absorption spectrum was measured at a concentration of 1.3mM in 50 mM Tris, 50 mM NaCI, 1mM DTT, 10 M zinc acetate, pH8.0, with the emperaturemaintained at 150 K using a Displexrefrigerator. Model compounds ZnS and Zn(Im),(C101)2 (referred toas Zn(Im),) were also measured in the fluorescence mode using thegermanium detector and a Stern-Heald type ionization chamber.hepreparation of the Zn(Im), model has been previously described (24).This model was measured at 150 K at a concentration of about 10mM. The ZnS was measured in thepowder form, sieved to 400 mesh,and spread uniformly on Scotch tape. At least 6 million si pal countsper data pointwere collected.EXAFSdata were reduced andanalyzed following establishedprocedures (23). A criterion for the east squares fitting results wasused to distinguish between the structural models as previously de-scribed 25). Briefly, the quality of a itcan he measured by thefollowing equation:

whereQ is the statusf thefit,nj is the combined phase and amplitudeerror for the tit at point i, and n$ is the experimental and modelingerror. Nand P are the independent data points (26)ncluded withinthe data range and the number f parameters used in the fit, respec-tively. The experimental and modeling error can he estimated fromthe variation of different measurements and from the modeling of aknown structure, respectively. A fit isaccepted if the status parame-ter, Q, is less than or equal to unity, and rejected otherwise. We alsoused the radial distribution function (RDF) method (27,28) to analyzethe first coordination sphere of ZnZ'. This method is not biased byassuming a structural model, and so provides an independent checkon the fitting results.Cysteine Deriuatization-The time course of E7 protein derivati-zation with ["Cliodoacetamide (["CI IAM) was nitiated by mixing110p l of E7 protein (0.7 mg/ml in 50 mM Tris, 50 mM NaCI. 1 mM

DTT, and0.1mM Zn acetate, pH 8.0) with 55 pl "C-IAM (5.8mCi/mmol; Amersham). A t various time points. 15pl of the mixture wasadded o 100 p of 8% trichloroacetic acid containing 0.S mg/rnlbovine serum albumin as a carrier. The precipitate was pelleted hycentrifugation, washed 4 times with I 50 pI o f 5" ; trichloroacetic acid,and dissolved in 100pI of 1 N NaOH. After neutralizatinn with 100pI of 1N HCI, the sample was diluted with scintillation cocktail andcounted. All data were ohtained in triplicate. The negative controlwas performed in an identical manner without E7 protein. Additionalexperiments were performed in the same huffer plus 6 SI kqnlanidineor 6M guanidine and 10mM EDTA.To analyze theE7 protein for the reartivitvof individrnlal cysteines.theE7protein WRS derivatizedwith odoacetnrnide ( I A M ) undernative and mildly reducing conditions. The purified E7 protein ( 5 rngat 0.1 mM E7 in 50 mM Tris, 50 mM NaCI. 1 mM 1 ) ' I T . 0. 1 mM zincacetate,pH = 8 .0 ) was diluted 1:l with an 11 rnM I AM stock (inwater) yielding ahout a 4-fold excess of IAM over free sulfhydql. Thereaction was allowed to proceed fnr 5 min, after which the derivnti-zation was terminated by gel chromatographv (G-'25)n degassed 1fH)mM Tris-CI , pH 8.0.The IAM-modified E7 protein was mmediately derivatized with4-vinylpyridine (4-VP) under denaturing conditions in the presenceof EDTA. Fifteen microliters of 4-VI' was addd to the desalted E7pool (final 4-VP] = 9 mM), then gtnlanidine-HCI and EDTA wereadded to final concentrations of 6 M and 10 mM. respectively. Thereaction proceeded for 1.5 h in the dark. Another gel fi ltration stepwas used to remove unreacted 4-VP.Derivatized E7protein wasdigestedwith trypsinor CNRr andpeptide fragments were isolated hy reverse phase HI'1,C on a VydakC18 column. All peaks containing greater than -5'; of the total lt.,l,,-absorhing material were collected and analyzed hy quantitative S -terminal amino acid sequencing.

RESULTSHPV-16E7protein was produced in . coli using the strongpromoter system of the pTAC"' vector (22). Initially, hisconstruct produced very littleE7protein. However, insertionof a 16-amino acid extension derived from theUC-18plasmidyielded high evels of an E7 fusion protein containing a 16-amino acid N- terminal extension. T he successful expression

vector, TACs"E7202, ispresented in Fig. 1 (pnnrl A ).T he high level of expression of E7 (Fig. l R , lanp I ) ed toan efficient purification scheme.A large percentage of the E7protein remained insoluhle after lysis, suggesting the deposi-tion of E7 into nclusion bodies. niti al attempts to run thesoluble E7 material onto the DEAE column resulted n theE7 elutingacross heentiresaltgradient. (100-400 mM).Addition of denaturingandreducingagents o he ysate,however, solubil ized most of the E7present in the cells (Fig.

6 2 -30 .\

\. 1'' dFIG. 1. Expression andpurification of HP V-1 6 E7protein.A , the HPV-I6 E7 coding sequence w as inserted hehind the pTA('promoter (12). The first 6 amino acids are derived from the cloningprocedure. I ?.SDS-polyacrylamide ge elertrophorrsis was performedby themethod of Laernmli (33) on n I G r i hisacrylarnide ge andstained withCoomassiedve. Nurnhrrs to the l ~ f t f gel representmigration positions of prestained markers of the indicated molrculitr

mass (in kDa). Imne 1. total E. coli cell lysate; lnnr 2. soluhilized I8M urea) ysate supernatant; lane 9 , DEAR-Sepharose column eluatepool; lane4 , Sephadex C-100column eluatepool.


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6912 Characterizationof HPV-16E7 Proteinl B , l ane 2 ) and yielded a sharp peak of E7 eluting from theDEAE column at approximately 250 mM NaCl (Fig. lB , l ane3).Early refolding attempts on the DEAE eluate resulted inhighly aggregated E7 protein. A wide range of refolding con-ditions were surveyed and screened for the production of lowmolecular weight E7 products on a calibrated HPLC sizeexclusion column (data notshown). Most conditions resultedin E7 species eluting at anapparent molecular mass of 60 or40 kDa or mixtures of the two. When high levels of reducingagent (glutathione) and low levels of ZnZ+and E7 were usedfor refolding, a mixture of 40- and 20-kDa species was ob-served. The 20-kDa species was unstable and was convertedto the 40-kDa form upon dialysis out of refolding buffer.Irrespective of the refolding conditions used, the E7 fusionprotein migrated with an apparent molecular mass of 17 kDaon an SDS-polyacrylamide electrophoresis gel, although theactual molecular mass of the protein s 12.5 kDa (Fig. lB ,lanes3and4 ) .The refolding conditions selected (see M aterials and M eth-ods) resulted in the 60-kDa species at concentrations above0.5 mg/ml and shifted to the 40-kDa species upon dilution(0.15 mg/ml). The 60-kDa species was purified on a G-100size exclusion column and utilized for subsequent character-ization (Fig. lB , l ane 4 ) . The identity of this protein wasverified by Western blot analysis, co-immunoprecipitationwith reticulocyte lysate-translated RB protein, and N-termi-nal amino acid sequencing of tryptic peptides. Atomic emis-sion studies indicated that the purified protein bound 1.04mol of zinc/mol of protein, confirming the zinc binding ob-servations of other groups (7, 21). No binding of purified E7to DNA-cellulose was observed in a buffer containing 25mMHEPES, 100 mM NaC1,5 mM MgC12, and 0.1% T ri ton X-100(data not shown).To determine the ligands of the protein-bound Zn2+,EXAFS studies were performed on the purified protein. TheEXAFS x ( k )data were reduced by background subtraction,E (energy) to k (electron wave number) conversion, andnormalization to an edge step, and are presented in Fig. 2A.The low frequency oscillations of the protein-bound Zn2+arein phase with those of ZnS, in which zinc is coordinated by 4sulfur atoms, indicating similarity in the zinc ligation. Thissimilarity is reinforced by comparison of the Fourier trans-forms for the two complexes, which are presented in Fig. 2B.Least squares fitting was performed on the isolated firstshell x(k)data between 2.5 and 10.519 in k-space to obtainquantitative results on the nature of the metal ligation in E7protein. With he 1.3-19 R-window used in the back-transform,the number of degees of freedom in the dataange is about 7(26). The fitting was initially conducted with three fi ttingparameters: coordination number, average interatomic dis-tance, and itsDebye-Waller factor (a measure of disorder) byassuming a single sulfur distribution. In addition, two subshellfits with both sulfur and nitrogen contributions were con-ducted with three to five fitting parameters. An excellent fitwas obtained with the single-sulfur model, shown in Fig. 3A.Inclusion of nitrogens in the coordination shell resulted ineither an unrealistically large Debye-Waller factor (0.0319)or a poor fit if the Debye-Weller factor was constrained. T he4 sulfur contributions were required to obtain a good fit.Theleast square fitting results are presented n Table I, alongwith their status, Q, according to the fi tting criterion. Thesingle-sulfur coordination model can be accepted according tothi s criterion, and other models can be rejected. This modelindicates 4 sulfur ligands located at an average distance of2.3319 from the Zn2+,with slightly less disorder than in ZnS.

wA )F I G. 2 XAFS data on HPV-16 E7 and ZnS. A , XAFS rawdata x(k) of the HPV-16E7 protein (solid line) and ZnS (dashedline). The data were plotted as k2x(k) .B, Fourier transforms of theHPV-16E7protein and ZnS. The transform was performed from1to 12.5 A-l on kzx(k).The first coordination shell was isolated byinverse Fourier transform with a window from10 t o 23 A.

0 1.5 2 2.5 3 3.5 4wA )FI G. 3 Comparisonof ligand models to XAFS data.A , leastsquares fitting results of the first coordination shell of the HPV-16E7 protein. The experimental data (solid lines) can be adequatelyfitted by a 4-sulfur distribution (dashed line) at a distanceof 233 A.B , radial distribution function of the first shell of the E7 protein(solid line) compared with the RDF of ZnS (dashed line) calculatedfrom the known structure. The distribution was artificially broadenedby a Gaussian factor of u: =0.01A2to reduce the truncation rippledueto lack of high k information.The RDF method was used to independently verify theleast squares fi tting results (27, 28). The radial distributionfunction of the firstshell of zinc in the HPV E7 protein was

generated in three teps: determining the amplitude ratio andphase difference between the first shell x data of the protein


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Characterization of HPV-16E7 Protein 6913TABLE

Least squares fitting results for thefirstshellof the E7bound zinc ionThe values in the bracket are the plus/minus errors of the lastdigit. The sulfur model is based on comparison to ZnS, while thesulfur and nitrogen model is based on comparison to ZnS andZn(Im),.

Model N R (A) u (A ) StatusSulfur 4S 2.33 (1) -0.001 ( 1) 0.8Sulfur and nitrogen 4S 2.33 ( 1) 0.000(1) 2.81 N 2.07 (6) 0.003 (2)

E:q+ 3 pE-sL3S-ACM S-ACMS-PE t -

FI G. 4. Sequential Cys derivatization scheme.The large circlerepresents the external boundary of a hypothetical protein. Cysteinethiols ( -SH)and disulfides ( -S-S-)outside the circle represent resi-dues exposed to the solvent; buried or metal-bound thiols are repre-sented by symbols inside the circle. Treatment of native protein withiodoacetamide (reaction 1 ) derivatizes only reactive exposed thiols,generating acetamido-Cys(S-ACM).Subsequent denaturation, reat-ment with EDTA, and reaction with 4-vinylpyridine (reaction 2 )derivatizes buried and metal-bound thiols with the pyridylethylmoiety ( P E ) .Disulfides are unaffected by either treatment. Subse-quent digestion of the protein with protease or CNBr (reaction 3)generates peptide fragments which are isolated by HPL C and sub-mitted for N-terminal sequencing, where acetamido-Cys and pyridy-lethyl-Cys can be independently quantitated.and ZnS, performing cumulant expansion of the ratio anddifference down to k =0,and, finally, generating the RDF byan inverse sine transform of the data.The RDF of the firstshell of the E7protein is compared with that of ZnS (whichiscalculated from the known structure) in Fig.3B.The ratioof the integrated area of the distribution between the HPVE7 protein andZnS isabout 1.03:1, yielding 4.1 sulfurs in hefirst shell of the protein-bound zinc. In accord with the linearsquares fit, the RDF method indicates the 4-sulfur distribu-tion is ocated2.33A from theZn2+on with a slightly smallerdisorder than ZnS.The EXAFS data unambiguously demonstrate that theE7-boundZn2+contains 4 sulfur igands. To determine which ofthe7 cysteines inE7contributed these igands, a biochemicalapproach was developed to probe the structural ocation andnature of the cysteine residues of the E7protein. We distin-guished between solvent-accessible, buried or metal-bound,and disulfide-associated cysteines by using two chemical tagsto sequentially modify the cysteine sulfhydryls (Fig.4). Undernative conditions only the exposed free sulfhydryls are rapidlymodified by odoacetamide, generating S-acetamido-Cys (29).Subsequent denaturation and metal removal followed by de-rivatization with 4-vinylpyridine tagsall remaining freesulfhydryls, generating S-pyridylethyl-Cys, while disulfide-associated cysteines remain unmodified. The modified proteinwas then digested with trypsin (five cleavage sites) or CNBr

(five cleavage sites) and the resulting peptides purified byHPLC. N-terminal sequencing identified the peptides andquantitated the amountf both cysteine modifications at eachposition. Thus, for each cysteine in the protein, the relativeamounts of S-acetamido-Cys and S-pyridylethyl-Cys reflectthe reactivity of the cysteinyl thiol, with S-acetamido-Cysrepresenting reactive, solvent-exposed residues, and S-pyri-dylethyl-Cys representing buried or metal-ligated residues.Underivatized cysteine, such as might arise as a part of adisulfide, cannot be efficiently detected by N-terminal se-quencing. However, inter-peptide disulfides would be readilydetected by the presence of multiple peptide sequences in asingle HPLC peak, as well as by other methods. For instancedisulfide-associated peptides can also be identified by runningreduced and nonreduced samples of the peptides on the HPLCcolumn, because disulfide-linked peptides will generate twopeaks under reducing conditions.To select the appropriate derivatization time to distinguishbetween solvent accessible and buried or metal-bound cys-teines, a timeourse of [4C]iodoacetamidemodification of E7was performed. External free sulfhydryls should react morereadily with IAM than buried or metal-bound cysteines. Thedenatured, EDTA-treated protein reached maximal modifi-cation within 4 min, which indicates the rate of reaction ofIAM with fully exposed cysteines (Fig. 5) . The amount ofradiolabel incorporated into the protein was the same in thepresenceorabsence of reducing agents, suggesting the absenceof disulfide bonds. A model tetrapeptide (N-acetyl-Tyr-Cys-( N- methyl - Tyr ) - Asp-am de)ith a single cysteine was alsoused as a substrate forysteine modification. Ina competitionreaction, IAM was approximately 20-fold more reactive than4-vinylpyridine. With IAM alone, the derivatization of thepeptide was also complete within a 5-min incubation (datanot shown). The reaction rates for native anddenatured,metal-bound E7 were biphasic, indicating the presence ofboth astand slow reacting residues (Fig. 5). The nativeprotein reached 20% of the total label incorporation in theinitial fast hase of the reaction, whereas the denatured metal-bound protein reached 40%. Based on these results, an initialreaction time of 5 min with IAM was chosen to selectively

-I

MinutesFI G.5. Derivatization of E7 protein with [4C]iodoaceta-mide. RecombinantE7protein was reacted with [4C]iodoacetamidein various different buffer systems. Aliquots were withdrawn,quenched with trichloroacetic acid, and harvested as described underMaterials and Methods.Error bars represent the standard deviationfor analysis of each time point in triplicate. Curve legends are asfollows: filled circles (O),6 M guanidine, 10mM EDTA, 1mM DTT,100 mM Tris, pH 8.0;filled triangles (A),6 M guanidine,1mM DTT,100 mM Tris, pH 8.0;open sqwres (O),100 mM Tris, 1mM DTT, pH

8.0; filled inuerted triangles(V),noprotein, 1mM DTT, 100mM Tris,pH 8.0.


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6914 Characterizationof HPV-16 E7 Proteinmodify the more reactive cysteine residues inthe nativeprotein.Using the scheme outlined above, wefound that the ysteineat position 24 wasmodified 86% by iodoacetamide undernative conditions (Fig. 6), indicating that CysZ4s in the freesulfhydryl form and exposed to the solvent. Cysteines con-tained in theCys-X-X-Cys putative zinc-binding motifs wereall well protected from the solvent under native conditions:Cys6', Cysel, and CysQ4 wereodified by IAM 33,33, and 19%,respectively (CysG1 couldnot be isolated). This is consistentwith the proposed coordination of the zinc ion through theseresidues. Cysteines at positions 59 and 68,which are nothighly conserved among the HPVs, displayed moderate reac-tivity towards IAM under native conditions.No substantive changes in the HPLC lution profile of thedigested protein were observed upon the addition of excessDTT. Inaddition, N-terminal aminoacid sequence data fromeach tryptic peptide fraction indicated the presence of onlyone predominant sequence in each isolated fraction. Takentogether, these observations suggest that there are no disul-fide-associated cysteines in the refolded E7 protein underthese conditions.

DISCUSSIONThe E6 and E7 proteinsf HPV-16 are strongly associatedwith the development of human cervical carcinomas. Withthe ultimate goal of identifying potential therapeutic targetsfor HPV infections, wepurified and characterized the HPV-16E7 protein. As a part of this analysis, we investigated thechemical state of each cysteine residue in recombinantlyexpressed E7 protein, since previous studies have indicatedthat many of the cysteine residues are critical for transfor-mation and transactivation activities.The high level of expression of E7 inE . coli afforded us astraightforward purification scheme. The final E7 preparationwas routinely greater than 99% pure, as determined by SDS

gel electrophoresis and reverse phase HPLC. By altering therefolding conditions slightly, different sized complexes of theE7 protein could begenerated, resulting in species that elutedfrom a calibrated sizing column at an apparent molecularmass of about 20, 40, and 60 kDa, possibly representingmonomeric, dimeric, and rimeric E7. Thenature of theinteraction leading to oligomerization is not apparent fromthe current studies, although metal-mediated dimerizationcannot be ruled out.TheE7protein from HPV infected cell lines has beenshown to coimmunoprecipitate with pRB (9, 10). We havetested the pRB binding activity of the E . coli-produced E7protein in solution using T24 cell lysates and inuitro trans-lated pRB from rabbit reticulocyte lysates. Both sources ofpRB were able to form complexes with the E7 protein (19).An E7-derived peptide, N- acetyl - E7(21- 29) - pept i deamide,was previously shown to inhibit 50% (IC5,,)of the E7-pRBinteraction at a concentration of 40 nM, whereas the purifiedE7 protein fromE. coli displayed an IC50 of 2nM (19). Theseresults indicate that the 7 fusion protein hasa 20-fold higheraffinity for pRB than the peptide derived from the bindingNH2" ...". CZ4 .....". C5&C59-X-C61 ..."... (-68 ..... cg1-x-x-c94 .... ".COOH

%ACM-CYS: 66% 33% 74% NO 48% 33% 19%%PE- CY3 14% 66% 26% ND 52% 66% 61%

FIG.6. Quantitationof derivatized cysteinesby N-terminalsequencing. Individual peptide fragments from digests of derivatizedE7protein were analyzed by quantitative N-terminalsequencing. Theamount of acetamido-Cys(ACM-Cys)and pyridylethyl-Cys(PE-Cys)were individually quantitated. Percentages were derived by dividingthe amount of each derivative by the sum of the two derivatives.ND,not determined.

domain under the conditions tested.The increased affinity ofthe E7 protein for pRB suggests that thepRB binding regionof E7eithercontainsadditionalcontactsites outside theE7(21-29) domain or that this region of the protein is held ina specific conformation necessary for high affinity binding topRB by the remaining E7 sequences. These observationssuggest that the urified E7 protein s assuming a biologicallyactive conformation.The purified protein was analyzed for metal content byatomic emission spectroscopy, and a1:1molar ratio of zinc toprotein was observed. Zinc ions can be bound through cysteineand histidine side chains, to form zinc finger structures. TheE7 sequence differs from the consensus sequence of theTFIIIA-type zinc finger in several respects, including the lackof conserved aromatic residues, the apparent se of 4 cysteinesinstead of 2 cysteines and 2 histidines to coordinate the metalion, and a central loop which is 3 times the length of loopscontained in most known zinc finger structures (20). Thesedifferences, the high degree of conservation of the cysteineresidues, and the bservation that a peptide from the carboxylterminus of E7 containing only two cysteines was able to bindzinc (7), led us to investigate the nature of the metal ligands

by EXAFS.Both least squares fitting and RDF methods were used foranalyzing EXAFS data. Although the RDF method has notbeen widely used, i t is complementary to the least squaresmethod because it is not biased by an assumed model. Bothmethods determined that the Zn2+ on in the E7 protein iscoordinated to 4 sulfur ligands with an average interatomicdistance of 2.33A. These results are consistentith an earlieranalysis of the adenovirus E1A protein (24). The disorder ofthe ligands in the E7 protein s slightly less than thedisorderin ZnS, which may reflect the shorter interatomic metal-ligand distance in the protein. The time course of iodoaceta-mide modification of nativeE7 clearly shows a biphasicreaction rate, indicating the presence of fast andslow reactingcysteine residues. It is assumed that the ast reacting speciesrepresent the solvent-accessible free sulfhydryls, while theslower reacting species are derived from the buried and zinc-coordinated cysteines, although other environmental effectsmay also contribute to the differential reactivity of the cys-teine thiols. The final amount of radioactive label incorpo-rated into the E7 protein was independent of the derivatiza-tion conditions, suggesting that theprotein may be undergo-ing "breathing" or transient unfolding and thereby brieflyexposing buried or metal bound sulfhydryl groups to thederivatization reagent. A biphasic reaction was observed evenunder.denaturing conditions in thepresence of zinc. Roughly40% of the cysteines were modified in the fast phase of thereaction, suggesting that 3 out of the 7 cysteines were highlyreactive under denaturing conditions. In the presence of de-naturant plus EDTA, all of the cysteines are rapidly deriva-tized.The observation that the ultimate amount of radiolabelincorporated into E7 was independent of the presence ofreducing agent suggests that none of the cysteines are involvedinstable covalent disulfides, whichwouldbe resistant toderivatization in the absence of reducing agents (29). HPLCand sequencing analysis of the proteolytic fragments alsoprovided no evidence for a stable disulfide bond in recombi-nant E7. One possible caveat, however, is that the purifiedprotein was stored n the presence of 1mM DTT, and theinitial derivatization reaction with IAM was also performedunder mildly reducing conditions in order to preserve theintegrity of the essential cysteines. Because the sensitivity ofprotein disulfides to reduction with exogenous disulfides is


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Characterization of HPV-16E7 Protein 6915sensitive to theocal environment of the cystine, it is difficultto predict the effectof low levels of exogenous thiol on nativedisulfides (30). Nevertheless, the apparent lack of cystine inE7 is not surprising, since the interior of a mammalian cell isahighly reducing environment and disulfides are rarely ob-served in nuclear or cytoplasmic proteins (31).The sequential derivatization and N-terminal sequencingscheme successfully delineates between reactive, solvent-ac-cessible sulfhydryls and internalor metal-ligated thiols. Ourresults indicate that CysZ4 ontains a solvent-accessible freesulfhydryl. This cysteine residue falls within the region ofhomology shared between the SV40 large T antigen, E1ADrotein or adenovirus. andHPV-16 E7protein. This site has

REFERENCES1. DeVilleirs, E. M. (1989)J . Virol. 63, 4894-49032. Baker, C. C., Phelps, W. C., Lindgren,V., Braun, M. J., Gonda,3. Munger, K., Phelps, W. C., Bubb,V., Howley,P.M .,and Schlegel,4. Phelps, W. C., Yee, C. L ., Munger, K., and Howley, P. M . (1988)5. Storey, A., Pim, D., Murray, A., Osborn, K., Banks, L. , and6. Seedorf, K., Krammer, G., Durst, D., Sahai, D., and Rowekamp,7. Rawls, J . A., Pusztai, R., and Green, M. (1990) J . Virol. 64,8. Phelps, W. C., Yee, C. L .,and Howley,P. M. (1988) Cell 53,539-

M. A., and Howley,P.M. (1987) J . Virol. 61,962-971R. (1989) J . Virol.63,4417-4421Cell 53,539-547Crawford,L . (1988)EMBO J . 7, 1815-1820W. (1985) Virology 145, 181-1856121-6129

pRB can occur.The other4 conserved cysteine residues, Cys5, Cys, Cysgl,and CysS4, ave been shown to be required for efficient trans-formation and transactivation ctivity by mutational analysis(16, 17). These4 cysteines form two Cys-X-X-Cys motifs formetal binding. Our findings demonstrate that 3 of thesecysteine residues for which data were obtained are well pro-tected from the solvent under native conditions and are notinvolved in disulfide bonds under the conditions tested. Basedon stoichiometric considerations, it is likely that these 4cysteines represent the slow reacting phase of the derivati-zation time course depicted in Fig. 5. The apparent lack ofreactivity of these cysteines in the absence of chelating agentsand theEXAFS data indicating4 sulfur ligands for the boundzinc ion suggest that these cysteines are involved in bindingZn2+The E7 sequence does not fit the general consensus se-quences of the various zinc finger structures usually associatedwith sequence-specific DNA binding activity, possibly sug-gesting an alternative role for this structure in this protein.If the zinc-binding domain of E7 is not involved in maintain-ing aDNA-binding motif, i t may be associated with maintain-ing the pRB-binding domain of E7 in the optimal conforma-tion, or in zinc-mediated dimerization (32).In summary, we have purified the HPV-16 E7 proteinexpressed in E . coli in sufficient quantity andpurity to beginchemical and physical studies. Because of this proteins highaffinity for pRB and its ability to bind Zn2+webelieve thestructure of the recombinantly expressed fusion protein ssimilar to that of the native virally produced protein. Thecysteine residue involved in pRB binding, Cyst, has beenunambiguously shown to be a solvent accessible free sulfhy-dryl available for interaction with pRB. EXAFSstudies dem-onstrate that the zinc ion is coordinated by 4 sulfur ligands,and our cysteine derivatization studies are consistent withmetal ligation through the4 cysteines contained in the Cys-X-X-Cys motifs. Additional studies will be required to dem-onstrate the functional role of this metal binding domain inthe E7 protein.

Acknowledgments-We express our gratitude to J . Bailey for cellpaste, to S.Thornton for atomic emission results, to J . Rodkey andT.Wood for sequencing data, and to thetaff of the NBPRT eamlineX9-A and the National Synchrotron Light Source.

105112. DeCaprio, J . A., Ludlow, J .W., Figge, J., Shew, J .-Y ., Huang, C.-M., Lee, W.-H., Marsillio, E., Paucha, E., and Livingston, D.M. (1988) Cell 54,275-28313. Schnider,J . F., Fisher, F., Goding, C. R., and Jones, N. C. (1987)14. Whyte, P., Williamson, N. M., and Harlow, E. (1989) Cell 56,15. Svensson, C., Bondesson, M., Nyberg, E., Linder, S., Jones, N.,16. Edmonds, C.,and Vousden, K. (1989) J . Virol.63,2650-265617. Storey, A., Almond, N., Osborn, K., and Crawford, L. (1990) J .Gen. Virol. 71, 965-97018. Watanabe, S., Kanda,T.,Sato, J., Fumno, A,, and Yoshiike, K.(1990) . Virol.64, 207-21419. Jones, R.E., Wegrzyn, R. J., Patrick, D. R., Balishin, N. L .,Vuocolo, G. A., Riemen, M. W., Defeo-Jones, D., Garsky, V.M .,Heimbrook, D. C., and Oliff, A. (1990) J . Biol. Chem. 265,

EMBO J . 6,2053-206067-75and Akusjarvi,G. (1991) Virology182,553-561

12782-1278520. Berg, J . M. (1989)Prog. Znorg. Chem. 37, 143-18521. Barbosa, M. S., Lowy, D. R., and Schiller, J . T. (1989) J . Virol.22. Linemyer, D. L., Kelly, L. J., Minke, J . G., Gimenez-Gallego,G.,Disalvo, J., and Thomas, K. A. (1987)BiolTechnology 5, 960-96523. Stern,E.A.,and Heald, S.M. (1986)inHandbook on SynchrotronRadiation (Koch, E., ed) pp. 955-1014, North-Holland, NewYork24. Webster, L. C., Zhang, K., Chance, B., Ayene, I., Culp, J . S.,Huang, W., Wu, F. Y ., and Ricciardi, R. P. (1991)Proc. Natl.Acad. Sci. U.S. A,, 88,9989-999325. Zhang, K., Stern, E. A., Ellis, F., Sanders-Loehr, ., and Shiemke,A. K. (1988)Biochemistry 27, 7470-747926. Lee, P. A., Citrin, P. H., Eisenberger, P., and Kincaid, B.M.(1981)Rev. Modern P hys. 53, 769-80627. Bouldin, C. (1984) Ph.D. thesis, University of Washington, pp.28. Crosier,E. , Rehr, J ., and Ingalls, R. (1987) inX- Ray Absorption:

Principles, Applications, Techniques of EXAF S, SEXAF S, andXANES (Koningsberger, D., and Prins, R., eds) pp. 443-571,John Wiley& Sons, New York29. Kellaris, K.V., Ware, D. K., Smith, S., and Kyte, J . (1989)Biochemistry 28,3469-348230. Creighton,T. E. (1983) inFunctions of Glutathione: Biochemical,Physiological,Toxicological, and Clinical Aspects (Larson, A.,Orrenius, S., Holmgren, A., and Manneruik, B., eds) pp. 205-213, Raven Press, New York31. Kosower, N. S.,and Kosower, E. M. (1978) Int. Rev. Cytol. 54,109-16032. Frankel, A. D., Bredt, D.S.,and Pabo, C.0. 1988)Science240,33. Laemmli,U. K. (1970)Nature 227,680-685

63,1404-1407

63-72

70-83

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