INFECTION AND IMMUNITY, May 1994, p. 2071-2078 Vol. 62. No. 50019-9567/94/$04.00+0Copyright ©C 1994, American Society for Microbiology

Assignment of Functional Domains Involved in ADP-Ribosylation andB-Oligomer Binding within the Carboxyl Terminus of

the SI Subunit of Pertussis ToxinKATHLEEN M. KRUEGER AND JOSEPH T. BARBIERI*

Department of Microbiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received 29 November 1993/Returned for modification 3 February 1994/Accepted March 1994

The roles of the carboxyl terminus of the Si subunit (composed of 235 amino acids) of pertussis toxin in theADP-ribosylation of transducin (Gt) and in B-oligomer binding were defined by analysis of two carboxyl-terminal deletion mutants of the recombinant Si (rS1) subunit: C204, which is composed of amino acids 1through 204 of SI, and C219, which is composed of amino acids 1 through 219 of SI. C204 was expressed inEscherichia coli as a stable, soluble peptide that had an apparent molecular mass of 23.4 kDa. In a linearvelocity assay, the specific activity of C180 was 2% and that of C204 was 80% of the activity displayed by rS1in catalyzing the ADP-ribosylation of Gt. In addition, C204 possessed catalytic efficiencies (kca,/Km) that were110% at variable Gt concentrations and 40% at variable NAD concentrations of those reported for rS1. Thesedata showed that the catalytic activity of C204 approached the activity of SI. C204 and C219 were unable toassociate with the B oligomer under conditions which promoted association of rS1 with the B oligomer.Consistent with these results, mixtures of C204 or C219 with the B oligomer did not elicit a clusteringphenotype in CHO cells, whereas rSI which had associated with the B oligomer was as cytotoxic as nativepertussis toxin. These data indicate that residues between 219 and 235 are important in the association of theSI subunit with the B oligomer. These data allow the assignment of functional regions to the carboxyl terminusof SI. Residues 195 to 204 are required for optimal ADP-ribosyltransferase activity, residues 205 to 219 linkthe catalytic region of SI and a B-oligomer-binding region of SI, and residues 220 to 235 are required forassociation of SI with the B oligomer.

Bordetella petlissis, the causative agent of whooping cough,binds to ciliated epithelial cells of the human respiratory tractand produces a variety of virulence factors, including pertussistoxin (PT; Mr, 105,890). PT is a member of a group ofbacterially produced exotoxins known as ADP-ribosylatingtoxins. These toxins catalyze the transfer of ADP-ribose fromNAD onto specific target proteins within eukaryotic cells. PTcatalyzes the ADP-ribosylation of the ox subunit of a subset ofthe heterotrimeric GTP-binding proteins (including Gi, G<,and G,), thereby disrupting signal transduction (29).

Structurally, PT is composed of six noncovalently boundpolypeptides named SI, S2, S3, S4, and S5 (present in a1:1:1:2:1 ratio). The enzymatic activity resides within the SIsubunit, while the remaining subunits form a complex knownas the B oligomer. The B oligomer is required for binding anddelivery of the SI subunit into susceptible cells (28). Because ofthe oligomeric structure of PT, the SI subunit is bifunctional inthat it associates with the B oligomer and catalyzes theADP-ribosyltransferase reaction.

Various residues and regions of the SI subunit (composed of235 amino acids) which have a role in substrate binding andcatalysis have been identified by chemical modification studiesand mutagenesis. Our laboratory has identified a region of theSI subunit, located between amino acids 195 and 219, whichwas required for high-affinity binding of Gt and appeared tohave a role in catalysis (9). To define this region further, C204,a deletion peptide composed of the amino-terminal 204 aminoacids of the S1 subunit, was produced and characterized. Weshow that C204 displays a catalytic activity that approaches that

* Corresponding author. Mailing address: Microbiology, MedicalCollege of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI53226. Phone: (414) 257-8412.

of recombinant SI (rS1), which localizes the entire ADP-ribosyltransferase activity of the SI subunit to the amino-terminal 204 residues of the SI subunit. These results, coupledwith the previously reported data discussed above, indicatethat residues located between amino acids 195 and 205 have arole in the high-affinity binding of Gt and in catalysis.

In addition to its enzymatic function, the SI subunit mustassociate with the B oligomer. Previous studies indicated thatthe carboxyl terminus was important in the association of theSI subunit with the B oligomer (1, 6). In this report, we showthat residues carboxyl terminal to amino acid 219 of the S1subunit are required for association of the SI subunit with theB oligomer.

MATERIALS AND METHODS

Reagents were purchased from Sigma Chemicals unlessotherwise stated. [32P-adenylate phosphate]NAD was pur-chased from DuPont-New England Nuclear. Enzymes used forDNA manipulations and sequencing were purchased fromNew England BioLabs, Inc., and United States BiochemicalCorporation, respectively. Dithiothreitol (DTT), urea, and eggalbumin were purchased from Boehringer Mannheim, Pierce,and Calbiochem, respectively. Bio-Gel P-100 polyacrylamidegel filtration resin and columns (0.7 by 10 cm) were purchasedfrom Bio-Rad Laboratories. Other chromatography resinsused for protein purification were purchased from PharmaciaLKB Biotechnology. Ammonium hydroxide and formaldehydewere from Mallinckrodt. Purified PT and B oligomer were giftsof Rino Rappuoli (Istituto Ricerche Immunobiologiche, Siena,Italy) and were stored as ammonium sulfate precipitates at4°C, centrifuged, and resuspended in 25 mM Tris-HCI (pH 7.6)containing 2 M urea prior to use. Transducin (Gt) was purified

2071

2072 KRUEGER AND BARBIERI

ptdlC180

ptdlC204 -tC,

ptdlC219FIG. 1. Schematics of the plasmid constructs used.

represents DNA encoding amino acids found in the pro(The white box represents DNA encoding the DT signaldark gray box represents promoter DNA sequences withwhile the light gray box and the solid lines represen

sequences. To the right of the schematic are the resi(subunit encoded on the respective plasmids.

from bovine retina tissue (J. A. Lawson Co., Lincpreviously described (9). Rabbit anti-C180 peptglobulin G (2) was purified on a protein A-Sephs(Pierce Chemical Co.). 12'I-labeled protein A wasthe chloramine T method. Plasmid manipulatioformed essentially as described in reference 20. Es474 and SK110 are protease-deficient strainslaboratory (8, 17).

Construction of plasmids. (i) ptdlC180. ptdlC1protein which contains the diphtheria toxin (Drquence, followed by a glycine residue (the first a

DT), followed by residues 1 through 180 of the Sl1). ptdlC180 was constructed by overlap PCR mutwith amplification of the DT leader sequence froiand the C180 structural gene from ptacCl80 (2).first amino acid of DT, was included in this constithe recognition site for cleavage by the leader pe

(ii) ptdlC219. ptdlC219 encodes a protein whichDT leader sequence, followed by a glycine residucresidues 1 through 219 of the Si subunit (Fig. 1).]constructed by replacement of the SalI-HindIIIptdlC180 (described above) with the correspondiment from ptacC219 (8).

(iii) ptdlC204. ptdlC204 encodes a protein wlthe DT leader sequence, followed by a glycinelowed by residues 1 through 204 of the Si subiptdlC204 was constructed as follows. A stopArg-204, followed by a HindlIl site, was eng

ptdlC219 (described above) by using a PCR protoSalI-HindIll fragment of ptdlC219 was replaccorresponding fragment of the above-describedgenerate ptdlC204. The nucleotide sequence was

DNA sequencing using the 7-deaza-dGTP kitStates Biochemical Corporation.

Subcellular fractionation of E. coli. Subcelluations were performed essentially as previously c

Overnight cultures of E. coli 474 containing eitheiptdlC204 were diluted into L broth containinampicillin per ml. Cultures were shaken at 2incubated at 30°C until they had reached an o0(A600) of approximately 1.0. An equivalent nur

(determined as equivalent optical density unitsculture was pelleted by centrifugation at 5,000 xat 4°C. Cells were resuspended in 500 RI of 40 n(pH 8.0) containing 30% sucrose. Lysozyme3-mg/ml solution in 0.1 M EDTA) was added, ansion was incubated on ice for 30 min. Magnesium

,ul of a 1.0 M solution) was added, and the sample wasResidues of Si centrifuged at 5,000 x g for 10 min to isolate the periplasmic1-180 fraction (soluble material). The particulate fraction (contain-

ing spheroplasts) was subjected to five cycles of freezingfollowed by thawing. The cytoplasm was isolated as the soluble

1-204 fraction after centrifugation at 16,000 x g for 15 min. Thepellet (containing the membranes and associated proteins) wasresuspended in 500 RI of 25 mM Tris-HCl (pH 7.6) containing0.1% sodium dodecyl sulfate (SDS). Equivalent volumes of

- 1-219 each fraction were subjected to SDS-polyacrylamide gel elec-

The black box trophoresis (SDS-PAGE) on a 12% gel. Peptides separated bycessed peptide. SDS-PAGE were either stained with Coomassie blue or trans-sequence.The ferred to nitrocellulose and subjected to Western blotting

iin the plasmid, (immunoblotting) (5). The nitrocellulose was incubated withIt vector DNA anti-C180 peptide immunoglobulin G, followed by125I-labeleddues of the SI protein A, as previously described (3).

E. coli transformed with ptdlC180 or ptdlC204 was alsoanalyzed for steady-state expression of C180 or C204. Cellswere centrifuged at 16,000 x g for 2 min and resuspended in

oln, Nebr.) as SDS sample buffer containing,B-mercaptoethanol. Samples:ide immuno- were subjected to SDS-PAGE as described above.arose column Purification of peptides. (i) rSl and C180. rSl was purifiedsprepared by from E. coli SK110 and C180 was purified from E. coli 474 astns were per- previously described (8).cherichia coli (ii) C204. C204 was purified from E. coli 474 containingused in this ptdlC204 as follows. Cells (2 liters) were harvested at an

optical density (A600) of 0.8 by centrifugation at 7,000 x g forL80 encodes a 20 min and resuspended in 30 ml of 40 mM Tris-HCl (pH 8.0)T) leader se- containing 30% sucrose. Lysozyme (2 ml of a 6-mg/ml solutionmino acid of in 0.1 M EDTA) was added, and the suspension was incubatedsubunit (Fig. on ice for 30 min. Magnesium chloride (2 ml of a 1.0 Mtagenesis (12) solution) was added, the sample was centrifuged at 17,000 x gm pDO1 (24) for 40 min, and the periplasmic fraction (soluble material) was. Glycine, the isolated. The periplasm was adjusted to 60% saturated ammo-ruct to define nium sulfate, incubated at 4°C overnight, and centrifuged atvptidase. 17,000 x g for 20 min. The pellet was resuspended in 25 mMicontains the Tris-HCl (pH 7.6) and chromatographed over Sephacryl, followed by S-200HR (550-ml column equilibrated in 25 mM Tris-HCl [pHptdlC219 was 7.6]). Fractions containing C204 were pooled and adjusted tofragment of 60% saturated ammonium sulfate. This chromatographic stepng gene frag- was sufficient to separate full-length C204 from other immu-

noreactive peptides and allowed the purification of 0.1 mg ofhich contains C204.residue, fol- (iii) C219. C219 was purified fromE. coli SK110 containingunit (Fig. 1). ptdlC219. The periplasmic fraction was isolated (as describedcodon after for C204), adjusted to 60% saturated ammonium sulfate,,ineered into incubated at 4°C overnight, and centrifuged at 17,000 x g for)col (13). The 20 min. The pellet was resuspended in 25 mM Tris-HCl (pH-ed with the 7.6) containing 10% saturated ammonium sulfate and chro-construct to matographed over a 5-ml phenyl Sepharose CL-4B column.confirmed by C219 was eluted by decreasing the ionic strength of thefrom United chromatography buffer to 1 mM Tris-HCl (pH 7.6). Fractions

containing C219 were pooled and chromatographed over aslar fraction- 5-ml DEAE-Sephacryl column. C219 was eluted with a linearlescribed (2). gradient of increasing NaCl concentrations (0 to 150 mMr ptdlC180 or NaCl). Fractions containing C219 were pooled, adjusted tog 0.1 mg of 60% saturated ammonium sulfate, and stored at 4°C.50 rpm and ADP-ribosylation of Gt. rSl, C204, and C180 were incubatedptical density for 10 min at room temperature in 50 mM Tris-HCl (pHnber of cells 7.6)-0.1 mg of egg albumin per ml-20 mM DTT prior to the,) from each assay for ADP-ribosyltransferase activity (17, 22).g for 10 min Linear velocity. Reaction mixtures contained 0.1 M Tris-nM Tris-HCl HCl (pH 7.6), 20 mM DTT, 0.02 mg of egg albumin per ml, 0.1(25 [lI of a JIM [ 2P-adenylate phosphate]NAD (specific activity was ad-d the suspen- justed to 250 Ci/mmol by addition of unlabeled NAD), 0.1 ,uMchloride (25 Gt, and an aliquot of either C180, C204, or rSl. The reaction

INFECT.IMMUN.

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FUNCTIONAL DOMAINS OF THE Si SUBUNIT 2073

mixture was incubated at room temperature, and 20-p,l ali-quots were removed at 10, 20, 30, 40, 60, 80, and 100 min andeither subjected to precipitation with trichloroacetic acid oradded to SDS sample buffer containing ,B-mercaptoethanoland subsequently analyzed by SDS-PAGE. Incorporation of32p into the ca subunit of G, (Gt,) was determined andvelocities were calculated as previously described (16). Speci-fic activities (see Table 2) represent the linear portion of thecurve that measured the incorporation of 32p into trichloro-acetic acid-precipitable material versus time following normal-ization for the amount of rSl, C204, or C180 in the reactionmixture.

Michaelis-Menten kinetic analysis of C204-catalyzed ADP-ribosylation of Gt. (i) Variable Gt. Reaction mixtures (70 p,l)contained 0.1 M Tris-HCl (pH 7.6), 20 mM DTT, 0.02 mg ofegg albumin per ml, 0.1 PLM [32P-adenylate phosphate]NAD(specific activity was adjusted to 250 Ci/mmol by addition ofunlabeled NAD), a defined concentration of Gt, and an aliquotof C204. The final concentration of Gt was varied between 0.1and 1.9 ,uM by serial dilution of a stock of Gt (stored at - 20°Cin 40% glycerol) into 40% glycerol prior to addition to thereaction mixture. At each concentration of Gt examined, 20-,ulaliquots of the reaction mixture were removed at 20, 40, and 60min, added to 6 ,lI of SDS sample buffer containing ,B-mercap-toethanol, and boiled for 5 min. Samples were subjected toSDS-PAGE on 12% gels. Incorporation of 32p into Gt, was

determined, and velocities were calculated as previously de-scribed (16). The rate of ADP-ribosylation of Gt was linear,and less than 10% of the available NAD and Gt was utilized.Data were subjected to Michaelis-Menten kinetic analysis withthe assistance of Enzfitter (nonlinear regression analysis soft-ware written by R. J. Leatherbarrow and distributed byElsevier, Cambridge, United Kingdom). The values presented(see Table 2) are averages of two independent experiments.

(ii) Variable NAD. Assays were performed as described inthe previous paragraph, with the following exceptions. Theconcentration of G, was held constant (0.5 ,uM), while theconcentration of NAD was varied between 3.9 and 44 ,uM. Foreach concentration ofNAD assayed, a pool containing 90 Rd ofthe reaction mixture was made. Aliquots (20 Ild) of the reactionmixture were removed from each pool after 20, 40, 60, and 80min and assayed for incorporation of radiolabel into Gta. At allconcentrations ofNAD tested, the rate of ADP-ribosylation ofGt was linear and '<10% of the available NAD and Gt wasutilized.

Association of C204, C219, or rSl with the B oligomer. rSl,C219, or C204 (approximately 22 pmol) and the B oligomer(approximately 12 pmol) were incubated for 1 h at roomtemperature in 25 mM Tris-HCl (pH 7.6) containing 1 M ureain a total volume of 50 p,l. These conditions have previouslybeen shown to promote association of the S1 subunit with theB oligomer (3, 4, 6, 27). The mixture was then subjected toBio-Gel P-100 gel filtration (see below).

Protease cleavage of PT. Reaction mixtures (120 RI) con-

tained 0.1 M Tris-HCl (pH 7.6), 0.15 ,uM PT, and 15 pLg ofchymotrypsin per ml. The reaction mixture was incubated atroom temperature for 15 min prior to addition of 0.3 mg oftrypsin inhibitor per ml. A 50-,ul aliquot of the reaction mixturecontaining 7.5 pmol of chymotrypsin-treated PT was subjectedto Bio-Gel P-100 gel filtration (see below).

Bio-Gel P-100 gel filtration. Reaction mixtures were chro-matographed on a Bio-Gel P-100 gel filtration column (0.7 by10 cm; 3.5 ml of resin) which had been equilibrated in 25 mMTris-HCl (pH 7.6) containing 150 mM NaCl. Fractions (100 RI)were collected, dried under vacuum in a centrifuge, resus-

pended in 35 [lI of SDS sample buffer containing 1-mercapto-

TABLE 1. Subcellular localization of C180 and C204 in E. coli

Subcellular fraction (%)aPeptide

Periplasm Cytoplasm Membranes

C180 46 26 28C204 48 15 37

a Data were quantitated from autoradiograms of subcellular fractionations ofC180 and C204 (see Materials and Methods) which were subjected to AMBISoptical densitometry. C180 present in the cytoplasm and membrane fractionscontained immunoreactive material which migrated at a position correspondingto that of material containing an uncleaved signal sequence. This material wasincluded in the quantitation. C204 was found as a single immunoreactive peptide.The percentage of the total amount of each peptide present in the three fractionsis shown.

ethanol, and boiled for 7 min. Aliquots (15 ,u) were subjectedto SDS-PAGE on 12% gels. Proteins were visualized by eithersilver staining (10) or Western blotting (5) with Si subunit-specific antiserum. Recoveries from the column were approx-imately 150%, as previously discussed (16).

Quantitation of peptides present in Bio-Gel P-100 columneluant. SDS-PAGE gels were subjected to Western blottingfollowed by autoradiography to visualize the Si subunit or itsderivatives (rS1, C219, C204, and the chymotryptic Si pep-tide). Relative amounts (referred to as net counts) of the Sisubunit were quantitated by AMBIS optical densitometry ofthe autoradiograms. The S1 subunit in each fraction wasreported as a percentage of the total [100 x (net counts inan individual fraction/sum of net counts in all fractions as-sayed)].SDS-PAGE gels were subjected to silver staining to visualize

the subunits of the B oligomer. The silver-stained band corre-sponding to the S2, S3, or S4 subunit was analyzed by AMBISoptical densitometry. The amount of the respective subunitpresent in each fraction was reported as a percentage of thetotal as described in the previous paragraph.

Quantitation of PT, rSl, deletion peptides, and Gt. PT, rSl,C219, C204, and C180 were subjected to SDS-PAGE on gelscontaining a known amount of a C180 peptide standard (2).Gels were stained with Coomassie blue and subjected toAMBIS optical densitometry to calculate the quantity of thepeptide present. The concentration of ADP-ribosylable G, wascalculated as previously described (16).

Chinese hamster ovary (CHO) cell clustering. rSl, C219, orC204 was incubated with the B oligomer as described above.Fourfold serial dilutions of PT or the above-described mixtureswere made in 25 mM Tris-HCl (pH 7.6) containinp 1.0 mg ofalbumin per ml. Aliquots were added to 1 ml of 10 CHO cellscultured in HAMS F-12 medium supplemented with 10%newborn calf serum. After 24 h of incubation, cells wereobserved microscopically for clustering morphology (11).

RESULTS

Expression and subcellular localization of C219 and C204.(i) C219. C219 was expressed in E. coli SK110. Replacement ofthe S1 leader sequence with the DT leader sequence yielded a10-fold increase in the steady-state expression of C219 and a2.5-fold increase in the amount of C219 localized to theperiplasm (data not shown).

(ii) C204 versus C180. Subcellular fractionation of C204versus C180 revealed that the same percentage (50%) of bothpeptides was localized to the periplasm (Table 1). Approxi-mately half of the immunoreactive material found in the

VOL. 62, 1994

2074 KRUEGER AND BARBIERI

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FIG. 2. SDS-PAGE of C219, C204, and C180. Duplicate samples ofC219, C204, and C180 were added to SDS sample buffer containingeither 3-mercaptoethanol or no reducing agent (labeled Reduced or

Oxidized, respectively). After the samples were boiled for 5 min, theywere subjected to SDS-PAGE on a 12% gel. The gel was subsequentlystained with Coomassie blue. The left lane contained molecular mass

markers. The respective masses are listed in kilodaltons.

cytoplasm and membrane fractions of the C180 peptide pos-

sessed an apparent molecular mass corresponding to C180containing an uncleaved signal sequence. All of the C180found in the periplasm appeared to have had the signalsequence cleaved off. The immunoreactive material found inall three fractions of C204 migrated with the same apparentmolecular mass (data not shown).

Purification of C219 and C204 and migration of the peptideson SDS-PAGE. (i) C219. Multiple chromatographic steps(including gel filtration and hydrophobic and anion-exchangechromatographies) were successful in separating C219 fromother E. coli proteins but unsuccessful in separating C219(apparent molecular mass, 26.2 kDa) from a probable degra-dation product of C219, an immunoreactive peptide with an

apparent molecular mass of 21.9 kDa. These proteins were thepredominant bands detected when C219 was subjected toSDS-PAGE in the presence of a reducing agent (Fig. 2). In theabsence of a reducing agent, three predominant immunoreac-tive, as well as Coomassie-stainable, bands were detected with

corresponding molecular masses of 24.1, 26.2, and 47.4 kDa.The 47.4-kDa band may be a dimer formed from the 24.1-kDapeptide by an intermolecular disulfide bond.

(ii) C180. When purified C180 was subjected to SDS-PAGEin the presence of a reducing agent, a single peptide whichmigrated with an apparent molecular mass of 20.2 kDa was

detected. In the absence of a reducing agent, two bands whichmigrated with apparent molecular masses of 21.6 and 43.7 kDawere detected (Fig. 2). The 43.7-kDa band most likely repre-

sents dimers of the 21.6-kDa species formed as the result of anintermolecular disulfide bond between the single Cys (Cys-41)in C180, since both bands were recognized by anti-C180immunoglobulin G (data not shown).

(iii) C204. The C204 peptide was purified by isolation of theperiplasm from cells containing ptdlC204 and subsequent gelfiltration. When purified C204 was subjected to SDS-PAGE,either in the presence or in the absence of a reducing agent, a

single immunoreactive, as well as Coomassie blue-stainable,peptide which migrated with apparent molecular masses of23.4 and 23.0 kDa, respectively, was detected (Fig. 2).The C204 peptide was subjected to amino-terminal se-

quence analysis. The resulting sequence, Gly-Asp-Asp-Pro-Pro-Ala-Thr-Val-Tyr-Arg, was identical to the amino-terminalsequence of the Si subunit, with the exception of an additionalGly residue at the amino terminus. This residue is the firstamino acid of DT and was introduced by the construction ofptdlC204 (see Materials and Methods). These data indicatethat the DT leader sequence was recognized and processed inE. coli.

C204-catalyzed ADP-ribosylation of G,. Linear velocities forrSl-, C204-, and C180-catalyzed ADP-ribosylation of G, were

determined at 0.1 ,uM NAD and 0.1 pFM G,. As shown in Table2, the specific activity of C204 was 80% of that of rSl and50-fold greater than that of C180. Similar results were ob-served when the assays were repeated.

Similar to rSl-mediated ADP-ribosylation of G, (6, 17, 23),the presence of the reducing agent DTT was required forC204-mediated ADP-ribosyltransferase activity and the activ-ity of C204 was not influenced by ATP. However, unlike otherpeptide derivatives of the S1 subunit expressed in our labora-tory, the lag observed prior to achievement of a linear velocitywas not eliminated by 10 min of preincubation of C204 with 20mM DTT (data not shown).C204 was subjected to Michaelis-Menten kinetic analysis

(Table 2). At 0.1 puM NAD and at variable Gt concentrations,C204 possessed a KmappGt of 0.9 pFM and a kcat of 7.5 h-1.These were within onefold of the values obtained underidentical conditions for rSl-mediated ADP-ribosylation of G,

TABLE 2. rSl-, C204-, and C180-catalyzed ADP-ribosylation of GtVariable G," Variable NAD'

Peptide Sp act' Mean KmappG, Mean kc,,, k.,,,/k,,," Mean Mean k,.a, k..,Ik.(A.M) ± SEM (h ) ± SEM ration (kMa ± SEM SEM ratio

C204 47 ± 3 0.9 ± 0.1 7.5 ± 0.9 8.3 10.2 ± 2.4 2.1 ± 0.1 0.2rSl 60 ± 3 0.6e 4.7e 7.8 20f 10 0.5C180 1.0 ± 0.1 NDI ND 16' O.if 0.01

a The specific activity is the mean linear velocity (millimoles of ADP-ribosylated G, per minute per mole of enzyme) ± the standard error of the mean.b NAD concentration, 0.1 pLM.' G, concentration, 0.5 ,uM.d Calculated for the average K,,, and k,a, values.e From Krueger and Barbieri (16).f From Cortina et al. (9).g ND, not determined.

INFECT. IMMUN.

FUNCTIONAL DOMAINS OF THE SI SUBUNIT 2075

(16). At 0.5 ,uM G, and with variable NAD concentrations,C204 possessed a KmappNAD of 10.2 ,uM and a kca, of 2.1min-. The Kmap1pNAD was within 1-fold of that observed forrSl and C180, while the kcat was 20-fold higher than the valuereported for C180 and 5-fold less than the value reported forrSl (9). The catalytic efficiency (kcatlK&) of C204 at variable G,concentrations was 110% of that of rS1, and at variable NADconcentrations it was 40% of that of rSl. These data showedthat the catalytic activity of C204 approached that of Si.

Association of rSl, C219, or C204 with the B oligomer. rSl,C219, or C204 was incubated with the B oligomer in thepresence of 1 M urea, conditions which have previously beenused to reconstitute PT from the B oligomer and rSl (3, 4, 6,27). Each mixture was subjected to Bio-Gel P-100 gel filtrationto determine if C204, C219, or rSI had associated with the Boligomer. The buffer used for the gel filtration (25 mMTris-HCl [pH 7.6] containing 150 mM NaCl) affords optimalseparation of PT from the SI subunit and does not inhibit theADP-ribosyltransferase activity of either PT or S1 (16).

Gel filtration of the B oligomer and rSl separately showedthat the B oligomer eluted primarily in the 1.5- to 2.3-mlfractions while rSI eluted in the 1.6- to 2.3-ml fractions. WhenrSI was incubated with the B oligomer and then subjected toBio-Gel P-100 gel filtration, the elution of the B oligomershifted to the 1.1- to 1.6-ml fractions (Fig. 3B, @). Two peaksof rSl were observed, one corresponding to the position ofelution of rSl alone (1.6- to 2.3-ml fractions) and anothercorresponding to the position of the elution of PT (1.1- to1.6-ml fractions) (Fig. 3A, 0). Aliquots of the column eluatewere also subjected to ADP-ribosyltransferase assays per-

formed either in the absence or in the presence of ATP (16).The 1.1- to 1.6-mi fractions possessed ADP-ribosyltransferaseactivity only when ATP was included in the assay mixture,whereas the 1.7- to 2.3-ml fractions possessed ADP-ribosyl-transferase activity independent of ATP (data not shown).ADP-ribosyltransferase activity of rSl is independent of ATP(7, 16, 23), whereas the activity of PT is stimulated by ATP (14,16, 18, 21, 23, 30).

Native PT and the 1.3- and 1.9-ml column fractions from thechromatography of rSl incubated with the B oligomer were

tested for the ability to cluster CHO cells (11). With respect tothe amount of rSl or Si present, the 1.3-ml fraction possessedcytotoxicity equivalent to that of native PT, while the 1.9-mlfraction did not cluster the CHO cells. The data presentedabove are consistent with the formation of reconstituted PT(rPT) upon incubation of rSI with the B oligomer.When either C219 or C204 was incubated with the B

oligomer and subsequently subjected to Bio-Gel P-100 gelfiltration, the B oligomer eluted in the 1.5- to 2.3-ml fractions,corresponding to the free B oligomer (Fig. 3B,* and V,respectively). In addition, both C219 and C204 eluted in the1.6- to 2.3-ml fractions, independently of whether they were

incubated alone or with the B oligomer prior to gel filtration(Fig. 3A,

*

and V, respectively). Mixtures of rSl, C219, or

C204 which had been incubated with the B oligomer were alsotested for the ability to cluster CHO cells. Although as little as

0.04 pmol of rSI which had been incubated with the Boligomer produced CHO cell clustering, no clustering was

observed when either 1.0 pmol of C219 or C204 which hadbeen incubated with the B oligomer was assayed. Together,these data indicate that C219 and C204 are unable to associatewith the B oligomer under these assay conditions.

Bio-Gel P-100 gel filtration of chymotrypsin-treated PT. Wehave previously shown that cleavage of PT with chymotrypsinresulted in preferential cleavage of the Si subunit at Trp-215(17). We subjected chymotrypsin-digested PT to Bio-Gel P-100

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IAI* rSl* C219v C204

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FIG. 3. Association of rSl, C219, and C204 with the B oligomer asdetermined by Bio-Gel P-100 gel filtration. Approximately 22 pmol ofrS1 (-),C219(-), or C204 (V) and approximately 12 pmol of the Boligomer were incubated at room temperature for 1 h in 25 mMTris-HCl (pH 7.6) containing 1 M urea. The material (50 pLl) wassubjected to Bio-Gel P-100 gel filtration (column size, 0.7 by 10cm, 3.5of ml resin) in 25 mM Tris-HCl (pH 7.6) containing 150 mM NaCl.The quantity of each individual peptide present in each fraction isreported as the percentage of the total [100 x (amount of peptide inan individual fraction/amount of peptide in all fractions assayed)] andwas calculated as described in Materials and Methods. The markers atthe top represent the void volume of the column (determined with bluedextran; molecular mass, 2 x 106 Da), the included volume (deter-mined with ATP), and the elution positions of two markers, lactoper-oxidase (molecular mass, 93 kDa) and carbonic anhydrase (molecularmass, 31 kDa). (A) The eluate was assayed for the presence ofS1-derived peptides. The brackets near the top represent the positionsof elution of rSl alone and theS1 subunit of PT. (B) The eluate wasassayed for the presence of the S2 subunit of the B oligomer (when theB oligomer was incubated with rSI prior to gel filtration) or the S4subunit of the B oligomer (when the B oligomer was incubated withC219 or C204 prior to gel filtration). The brackets near the toprepresent the positions of elution of the B oligomer alone and the Boligomer of PT.

gel filtration and analyzed the eluate for the presence of the Boligomer (determined by elution of the S3 subunit) and thechymotryptic Si peptide (composed of amino acids 1 to 215 oftheS1 subunit). Both the B oligomer and the chymotryptic Sipeptide eluted in the 1.1- to 1.7-ml fractions (Fig. 4, V and 0,respectively), corresponding to the elution of PT. These data

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8 [

2076 KRUEGER AND BARBIERI

0 c') -

> 0) C')

35

30

E-0E0

0

25

20

15

10

51.

0 -

1.0. . .

1.2 1.4 1.6 1.8 2.0 2.2 2.4 3.1ELUTION VOLUME (ml)

FIG. 4. Bio-Gel P-100 gel filtration of chymotrypsin-treated PT. PTwas incubated with chymotrypsin for 15 min as described in Materialsand Methods. The digestion was terminated by addition of a trypsininhibitor, and a 50-pl aliquot of the reaction mixture (containing 7.5pmol of PT) was subjected to Bio-Gel P-100 gel filtration. Thepositions of elution of the chymotryptic Si peptide and the B oligomer(determined by the position of elution of the S3 subunit) were

quantitated as described in Materials and Methods and are reported as

percentages of the total [100 x (amount of peptide in an individualfraction/amount of peptide in all fractions assayed)]. The brackets atthe top represent the positions of elution of rSI alone and the Sisubunit and B oligomer of PT (labeled PT). The markers at the toprepresent the void volume of the column (determined with bluedextran; molecular mass, 2 x 106 Da), the included volume (deter-mined with ATP), and the elution positions of two markers, lactoper-oxidase (molecular mass, 93 kDa) and carbonic anhydrase (molecularmass, 31 kDa).

indicate that the chymotryptic Si peptide did not dissociatefrom the B oligomer.

DISCUSSION

Mutagenesis of rSl has proved a useful approach to theidentification of the roles of various residues and regionsinvolved in ADP-ribosylation. While rSl was shown to possess

biochemical characteristics equivalent to those of Si purifiedfrom native holotoxin (8), most of the rSl was not soluble butwas associated with the membrane fraction in E. coli (3).Purification of active rSl was accomplished after solubilizationof rSl from the membrane fraction with 8 M urea. Oneconcern when performing mutagenesis of rSl was that theabsence of detectable enzymatic activity might not be due todisruption of a region involved in catalysis or substrate bindingbut rather be the result of improper folding upon removal ofthe urea. To circumvent this problem, soluble peptides havebeen engineered by deletion mutagenesis of the carboxylterminus of rSl to generate peptides composed of approxi-mately the first 180 amino acids of the Si subunit (1, 2).However, these peptides possessed reduced ADP-ribosyltrans-ferase activity compared with rSl (8, 19), which limits their

usefulness in kinetic studies. Isolation of a soluble peptidepossessing ADP-ribosyltransferase activity equivalent to thatof rSl would provide a useful tool for biochemical analysis.

Previous work in our laboratory indicated that C219, adeletion peptide composed of the first 219 amino acids of theSi subunit, was more soluble than rSl and displayed ADP-ribosyltransferase activity similar to that of rSl (8). However,purification of C219 proved difficult because of the apparentinstability of the peptide. In this report, C219 was separatedfrom other E. coli proteins but we were unable to separateC219 from apparent degradation products. Conversely, theC204 peptide, composed of the first 204 amino acids of the SIsubunit, was expressed as a stable peptide migrating with anapparent molecular mass of 23.4 kDa. Amino-terminal se-quence analysis indicated that the DT leader sequence hadbeen processed. Subcellular fractionation studies indicatedthat C204 was as soluble as C180. In addition to being a solublepeptide, C204 appeared to contain the single disulfide bondlocated within the SI subunit. Evidence to support this con-clusion includes the slight change in the migration of C204when it was subjected to SDS-PAGE in the absence of areducing agent, as well as the observation that C204 was devoidof enzymatic activity unless DTT was included in the reactionmixture.When assayed under linear-velocity conditions, the specific

activity of C204 was 80% of the ADP-ribosyltransferase activ-ity of rSl and 50-fold greater than that of C180. Michaelis-Menten kinetic analysis further indicated that at variable G,concentrations C204 possessed kinetic constants similar tothose reported for rSl. At variable NAD concentrations C204possessed a similar KmappNAD while the kc,t was 20-fold higherand 5-fold lower than the values reported for C180 and rSl,respectively. This kc,at difference between C204 and rSl may bedue to experimental variability between experiments thatassayed different G, preparations (17) or may reflect an actualdifference. Previous work in our laboratory indicated thatresidues between positions 195 and 219 of the Si subunit wererequired for efficient catalysis of the ADP-ribosyltransferasereaction (8). This region was required for high-affinity bindingof the Si subunit to the G, trimer and appeared to have a rolein the transfer of ADP-ribose to G, (9). The data presented inthis report indicate that C204 is capable of catalyzing theADP-ribosylation of G, with an efficiency that approaches thatof rSl. Together, these data further localize the region in-volved in the high-affinity binding of Gt to residues 195 through204 and imply that residues 205 through 235 are not directlyinvolved in ADP-ribosylation. We and other investigators haveidentified residues within the amino-terminal 180 residues ofSi that participate in the ADP-ribosylation reaction. From therecently solved crystal structure of PT (26), it appears thatresidues carboxyl terminal to residue 180 are not localizedwithin the proposed active site of Si. This suggests that theability of residues between positions 195 and 204 of Si toenhance ADP-ribosyltransferase activity is due to indirectalteration of the conformation of the active site. In addition,we have recently observed that the C180 peptide catalyzed theADP-ribosylation of a 20-amino-acid peptide that representsthe carboxyl-terminal residues of Gi,, at a rate similar to that ofrSl (9a), which showed that the entire catalytic machinery forADP-ribosylation resides with the first 180 amino acids of Si.Together, these results suggest that residues between 195 and204 do not constitute residues that are directly involved in theADP-ribosylation reaction mechanism but enhance ADP-ribo-sylation of the trimeric G protein via alignment of residueswithin either the active site of Si or the trimeric G protein foroptimal catalysis. The only residues within this region which

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FUNCTIONAL DOMAINS OF THE SI SUBUNIT 2077

contain potentially reactive side chains are Cys-201 and Arg-204. The remainder of the region is quite hydrophobic incharacter. Replacement of Arg-204 with Ser did not affect theADP-ribosyltransferase activity of SI or its toxicity whenassociated with the B oligomer (16a). Similarly, replacement ofCys-201 with Ser did not affect the ADP-ribosyltransferaseactivity of S1 (15). Together, these results indicate that thehydrophobic character of residues 196 through 204 is impor-tant for its function.C204 appears to be a useful peptide for analysis of products

engineered by site-directed mutagenesis. C204 is expressed ina soluble form that can be purified from the periplasm of E.coli and displays catalytic activity that approaches that of rSl.One disadvantage of using C204 as a target for mutagenesis isthe lag it displays before achieving a linear velocity. This lagwas not removed by preincubation with DTT under conditionswhich removed the lag displayed by other S1 derivatives.Because of the lag, the linear velocity at each substrateconcentration were calculated as the slope of the plot of 32pincorporated into G,at versus time rather than from a singletime point. PT also displays a lag before achieving a linearvelocity which is not removed by preincubation with DYT (16,21).

In addition to its enzymatic function, the S1 subunit mustalso interact with the B oligomer to display toxicity. Earlierstudies used nondenaturing gel electrophoresis to measure theassociation of SI with the B oligomer (4, 6, 27). In this study,Bio-Gel P-100 gel filtration was used to isolate rPT, formedfrom the association of rSI with the B oligomer. rPT possessedcytotoxicity equivalent to that of native PT, and like that ofnative PT, the ADP-ribosyltransferase activity of rPT wasstimulated by ATP (14, 16, 18, 21, 23, 30). Quantitation (basedon the amount of rS 1 eluting in the fractions corresponding tothe elution of PT) indicated that approximately 25 and 45% ofthe total rS1 and B oligomer present, respectively, had associ-ated to form rPT when these components were incubated at amolar ratio of 1.8 (rSl):1 (B oligomer). The advantages of gelfiltration over the nondenaturing gel electrophoresis systemused previously to determine if association had occurredinclude (i) the ability to view simultaneously free S1 and Siwhich has associated with the B oligomer and to quantitate theamount of each, (ii) the ability to purify free S1 from associ-ated SI, and (iii) the ability to subject the column eluant tofurther assay after gel filtration.We utilized Bio-Gel P-100 gel filtration to determine

whether deletion peptide C204 or C219 was capable of asso-ciating with the B oligomer. Previous studies have indicatedthat the carboxyl terminus of S1 played a role in the associationof the S1 subunit with the B oligomer. Burns et al. observedthat the S1 subunit which had been trypsin digested did notassociate with the B oligomer (6). Trypsin cleaved the S1subunit at the carboxyl terminus (6) after Arg-193 and Arg-181-182 (16a). Antoine and Locht found that replacement ofthe wild-type S1 gene with carboxyl-terminal truncations of theS1 gene (after residue 187 or 207) was insufficient to allowstable expression and secretion of S1 in B. pertussis, possiblybecause of a defect in the binding of the S1 subunit with the Boligomer (1). In this study, C204 and C219 (composed of theamino-terminal 204 and 219 amino acids of S1, respectively)were assayed for the ability to associate with the B oligomer.Neither C204 nor C219 was able to associate with the Boligomer under conditions shown to promote association ofrSI with the B oligomer. These data confirm the results ofprevious studies which indicated that the carboxyl terminusplays a role in the association of the S1 subunit with the Boligomer. Further, these studies indicate that residues between

219 and 235 are important for this association. Antoine et al.are currently mapping the interactions between S1 and the Boligomer in this region (18a). While neither C219 nor C204formed a stable complex with the B oligomer under theconditions tested, it is possible that other incubation conditionsexist that would allow C219 or C204 to form a stable complexwith the B oligomer.

These data allow us to assign functional roles to regionswithin the carboxyl terminus of the S1 subunit. Residues 195through 204 are required for optimal ADP-ribosylation oftrimeric G proteins. Amino acids 220 through 235 are hydro-phobic and are important for association of the S1 subunit withthe B oligomer. Amino acids 205 through 219 appear to linkthe catalytic core of the S1 subunit and the carboxyl-terminalB-oligomer-binding domain. These three functional assign-ments of residues within the carboxyl terminus of 51 areconsistent with the recently solved crystal structure of PT (26),which shows considerable structural homology to E. coli heat-labile enterotoxin (25). In the crystal structure of PT, residuesbetween 180 and 210 traverse the back of the active site of SI,residues between 210 and 227 include an unresolved regionwhich corresponds to the protease-sensitive region that waslocalized between 215 and 218 of S1 (17), and residuesbetween 228 and 235 constitute one region of S1 that interactswith the B oligomer. Our functional analysis of S1 also showedthat following cleavage of S1 at W-215, the amino-terminalportion of SI (residues 1 to 215) did not dissociate with the Boligomer, which suggested that residues amino terminal to 215interact with the B oligomer either directly or indirectlythrough interaction with residues 216 to 235. The crystalstructure of PT is consistent with this functional analysis andshowed several interactions between the amino-terminal re-gions of S1 and the B oligomer.

Future studies will define relationships between the func-tional properties of PT and its crystal structure with respect topathogenesis and vaccine development.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grants Al-30162 and AI-01087 (J.T.B.) from the National Institutes of Health.Amino acid sequence analysis was performed in the shared research

facilities of MCW under the supervision of Liane M. Mende-Mueller.

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2. Barbieri, J. T., B. K. Moloney, and L. M. Mende-Mueller. 1989.Expression and secretion of the S1 subunit and C180 peptide ofpertussis toxin in Escherichia coli. J. Bacteriol. 171:4362-4369.

3. Barbieri, J. T., M. Pizza, G. Cortina, and R. Rappuoli. 1990.Biochemical and biological activities of recombinant SI subunit ofpertussis toxin. Infect. Immun. 58:999-1003.

4. Bartley, T. D., D. W. Whiteley, V. L. Mar, D. L. Burns, and W. N.Burnette. 1989. Pertussis holotoxoid formed in vitro with a genet-ically deactivated SI subunit. Proc. NatI. Acad. Sci. USA 86:8353-8357.

5. Burnette, W. N. 1981. 'Western blotting": electrophoretic transferof proteins from sodium dodecyl sulfate-polyacrylamide gels tounmodified nitrocellulose and radiographic detection with anti-body and radioiodinated protein A. Anal. Biochem. 112:195-203.

6. Burns, D. L., S. Z. Hausman, W. Lindner, F. A. Robey, and C. R.Manclark. 1987. Structural characterization of pertussis toxin Asubunit. J. Biol. Chem. 262:17677-17682.

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the Si subunit of pertussis toxin required for efficient ADP-ribosyltransferase activity. J. Biol. Chem. 266:3022-3030.

9. Cortina, G., K. M. Krueger, and J. T. Barbieri. 1991. Thecarboxyl-terminus of the Si subunit of pertussis toxin confers highaffinity binding to transducin. J. Biol. Chem. 266:23810-23814.

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15. Kaslow, H. R., J. D. Schlotterbeck, V. L. Mar, and W. N. Burnette.1989. Alkylation of cysteine 41, but not cysteine 200, decreases theADP-ribosyltransferase activity of the S1 subunit of pertussistoxin. J. Biol. Chem. 264:6386-6390.

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25. Sixma, T. K., S. E. Pronk, K. H. Kalk, E. S. Wartna, B. A. M. vanZanten, B. Witholt, and W. G. J. Hol. 1991. Crystal structure of acholera toxin-related heat-labile enterotoxin from E. coli. Nature(London) 351:371-377.

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27. Tamura, M., K. Nogimori, S. Murai, M. Yajima, K. Ito, T. Katada,and M. Ui. 1982. Subunit structure of islet-activating protein,pertussis toxin, in conformity with the A-B model. Biochemistry21:5516-5522.

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30. Watkins, P. A., D. L. Burns, Y. Kanaho, T.-Y. Liu, E. L. Hewlett,and J. Moss. 1985. ADP-ribosylation of transducin by pertussistoxin. J. Biol. Chem. 260:13478-13482.

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