Proc. Nati. Acad. Sci. USA Vol. 82, pp. 7188-7192, November 1985 Biochemistry Limited proteolysis of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica (bifunctional enzyme/protein domains/protozoa) EDWARD P. GARVEY AND DANIEL V. SANTI* Departments of Biochemistry and Biophysics and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 Communicated by Thomas C. Bruice, June 27, 1985

ABSTRACT The structure and activity of the bifunctional found to be a dimer of apparently identical subunits of Mr thymidylate synthase-dihydrofolate reductase (TS-DHFR) 56,000 (5). To date, there has been no direct evidence for from the protozoan parasite Leishmania tropica were examined either homology or lack of homology between L. tropica by limited proteolysis with five different endopeptidases. Each TS-DHFR and the two individual enzymes from any non- reaction resulted in a rapid, time-dependent loss of TS activity protozoan species. and no effect on DHFR activity. The proteolytic products were Limited proteolysis has been used to examine the structure examined by NaDodSO4/PAGE; each digestion produced a of a number of multifunctional proteins. Results of these fragment of apparent Mr 35,000, and three of the five studies have led to proposals for the arrangement of the digestions generated a fragment of Mr 20,000. Attempts to various functions on the polypeptide (6, 7) and have produced separate the fragments under nondenaturing conditions failed, evidence that many multifunctional proteins exist as a series suggesting that the proteolyzed protein remains a dimer with of separate, independent domains (each domain reflecting a the gross structure ofthe subunits more or less undisturbed. In function) (7-9). We undertook the limited proteolysis of L. contrast, kinetic data indicate that some aspects of higher- tropica TS-DHFR in hopes of separating the two activities as order structure in the native protein are affected by proteolysis. proteolytic fragments, the size of each approximating their The fragments (Mr 36,600 and 20,000) generated by non-protozoan counterparts (i.e., a TS fragment of Mr Staphylococcus aureas V8 protease were subjected to sequence 35,000 and a DHFR fragment of Mr 20,000). We report analysis. Whereas neither the native protein nor the Mr 36,600 here the results of the limited proteolysis of L. tropica fragment yielded an NH2-terminal amino acid, we obtained the TS-DHFR. We propose an arrangement of the enzymatic sequence of the first 28 amino acids of the Mr 20,000 fragment. activities within the TS-DHFR polypeptide and suggest a This sequence bore strong homology with sequences situated structural model of the region that is selectively cleaved by within TS of human, Lactobacillus casei, Escherichia coli, and five different endopeptidases. bacteriophage T4. These and other data indicate that the TS-DHFR polypeotide consists of a DHFR sequence at the EXPERIMENTAL PROCEDURES blocked NH2-terminal and a TS sequence at the COOH- Enzymes, Antisera, and Activity Measurements. The bi- terminal end of the protein. The region that is the target of the functional TS-DHFR from 10-propargyl-5,8-dideazafolate- five proteases corresponds to a highly variable region within the resistant L. tropica (selection to be described elsewhere) was sequences of the other four TSs. We suggest that an insertion purified to homogeneity by methotrexate-Sepharose CL-6B occurs within the TS-DHFR sequence, positioned on the chromatography, as described (5). L-1-Tosylamido-2-phen- surface of the protein and quite vulnerable to the action of ylethyl chloromethyl ketone (TPCK)-treated trypsin, N-p- endopeptidases. tosyl-L-lysine chloromethyl ketone (TLCK)-treated a- chymotrypsin, Staphylococcus aureus V8 protease, Strepto- Thymidylate synthase (5,10-methylenetetrahydrofolate: myces griseus type XIV protease, elastase, and soybean dUMP C-methyltransferase, EC 2.1.1.45) and dihydrofolate trypsin inhibitor were purchased from Sigma. Rabbit antise- reductase (5,6,7,8-tetrahydrofolate:NADP' oxidoreductase, rum to L. tropica TS-DHFR was obtained after subcutaneous EC 1.5.1.3) catalyze consecutive reactions in the de novo injection of 150 ,Ag ofpure TS-DHFR mixed 1:1 (vol/vol) with synthesis of dTMP. In sources as varied as bacteriophage, Freund's complete adjuvant, followed 3 weeks later by a bacteria, and vertebrates, these two enzymes exist as distinct booster injection of 100 k&g of TS-DHFR mixed 1:1 with and readily separable enzymes (for reviews, see refs. 1 and Freund's incomplete adjuvant. Rabbit antiserum raised 2). In contrast, a bifunctional protein, thymidylate synthase- against Escherichia coli TS was provided by F. Maley (New dihydrofolate reductase (TS-DHFR), has been identified in a York State Department of Health, Albany); rabbit antiserum number of genera of protozoa which span a diverse group of against E. coli RT500 DHFR was provided by D. Baccanari the subkingdom (3, 4). This protein ranges in molecular (Burroughs Wellcome, Research Triangle Park, NC). Goat weight from about 110,000 to 140,000, with subunits of anti-rabbit Ig antibody-alkaline phosphatase conjugate was molecular weight 55,000-65,000. As has been noted (3-5), from Boehringer Mannheim. the subunit size of the protozoan TS-DHFR is close to the DHFR activity (10) and TS activity (11) were determined sum of the subunit size of TS (Mr 35,000) and DHFR (Mr spectrophotometrically at 250C. Also, DHFR was quantitat- 20,000) found in most other sources, suggesting that the ed by binding to [3H]methotrexate (1), and TS Was quanti- gene encoding TS-DHFR may have resulted from the fusion tated by binding to 5-fluoro-2'deoxy[3H]uridylate [3H]- of independent TS and DHFR genes. TS-DHFR from a FdUMP and (±)-5,10-methylenetetrahydrofolate (CH2H4- methotrexate-resistant cell line of the protozoan parasite folate) (12), as previously described. Leishmania tropica has been purified to homogeneity and Abbreviations: TS, thymidylate synthase; DHFR, dihydrofolate The publication costs of this article were defrayed in part by page charge reductase; FdUMP, 5-fluoro-2'-deoxyuridylate; CH2H4folate, (+)- payment. This article must therefore be hereby marked "advertisement" 5,10-methylenetetrahydrofolate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7188 Downloaded by guest on September 25, 2021 Biochemistry: Garvey and Santi Proc. Natl. Acad. Sci. USA 82 (1985) 7189

Table 1. Summary of limited proteolysis of L. tropica TS-DHFR t112, min Loss of enzymatic activity Loss of Mr 56,000 Predominant fragments Protease Specificity* TS DHFR subunitt generated,t M, x i0-3 S. aureus V8 protease Glu, Asp 18 No loss 40 37.3 - 36.6t (225 min) 20 Trypsin Arg, Lys 6 No loss 16 34.5 33.1 -* 31.6 (200 min) 21 -* 19 a-Chymotrypsin Tyr, Trp, Phe, Leu 20 No loss 35 Quartet, -37 (90 min) 19 Elastase Uncharged, nonaromatic 5 No loss 10 35 (135 min) mns§ S. griseus type XIV protease Nonspecific 10 No loss 14 36 (200 min) mns§ *Nature of amino acid residues donating carbonyl portions of susceptible peptide bands. tDetermined by NaDodSO4/PAGE. tArrows denote fragment generated from previous fragment during course of reaction. §Multiple nonspecific bands.

Gel Electrophoresis. NaDodSO4/PAGE (10-15% poly- RESULTS acrylamide) was performed essentially as described by The bifunctional TS-DHFR from L. tropica was subjected to Laemmli (13). Nondenaturing PAGE (12.5% polyacrylamide) limited proteolysis by use offive different endopeptidases: S. was performed according to Davis (14). Proteins were trans- aureus V8 protease, trypsin, a-chymotrypsin, elastase, or S. ferred from polyacrylamide gels to nitrocellulose filters (15) griseus type XIV protease (Table 1). Each of the five and probed with antibody (16); the alkaline phosphatase proteolytic reactions was monitored for enzymatic activities; conjugate was assayed as described (17). Peptides were in each digest there was a relatively rapid, time-dependent isolated from polyacrylamide gels for peptide mapping (18) inactivation of TS, which followed apparent first-order ki- and for NH2-terminal sequence analysis (19). netics for at least two half-lives (t1l2 < 20 min), and, under the Limited Proteolysis. TS-DHFR (0.1-0.5 mg/ml) in 50 mM conditions used, no loss ofDHFR activity. Curiously, in each Tes, pH 7.4/2 mM dithiothreitol/1 mM EDTA/5% (vol/vol) of the five digests, the rate of TS inactivation was approxi- glycerol was digested with endopeptidase (1:100 ratio, wt/wt) mately twice the rate of proteolysis (Table 1); when TS was or exopeptidase (5-10:100 ratio) at 25°C. To monitor enzy- completely inactivated, -50% of the Mr 56,000 subunit matic activities, aliquots (2-10 ,ul) were added directly to 1-ml remained intact. The extent ofproteolysis was also measured assay solutions. DEAE-Sepharose chromatography was as by assaying the ability ofTS or DHFR to bind their respective reported (5); the bound, proteolyzed TS-DHFR was eluted stoichiometric inhibitors, [3H]FdUMP or [3H]methotrexate. with a linear 0-0.2 M KCI gradient. Methotrexate-Sepharose V8 protease caused a time-dependent loss in the ability of TS chromatography was as described (5), except for the follow- to bind [3H]FdUMP and CH2H4folate, with the rate of loss ing additional washes of the bound, proteolyzed TS-DHFR occurring approximately twice as slowly as the rate of loss in before elution: 0.1% Triton X-100/10 mM potassium phos- catalytic activity (41/2 35 min vs. 18 min) and at approxi- mately the same rate as proteolysis (t1/2 - 35 min vs. 40 min). phate, pH 7.0, and 0.1% Nonidet P-40/10 mM potassium When TS-DHFR was digested with trypsin, there was no loss phosphate, pH 7.0. Enzyme was eluted with 1 mM 7,8- in the ability of DHFR to bind methotrexate; instead, the dihydrofolate in 50 mM Tes, pH 7.4/2 mM dithiothreitol/l amount of methotrexate bound to DHFR increased 2.3-fold mM EDTA. Sephadex G-150 (40-120 ,um; 14 x 56 cm after proteolysis was complete (that is, after >95% of the Mr column) was equilibrated and analysis was performed with 50 56,000 subunit had disappeared, as visualized by NaDod- mM Tes, pH 7.4/2 mM dithiothreitol/l mM EDTA/10% S04/PAGE). The kinetic Ki for methotrexate inhibition of (vol/vol) glycerol. In each chromatographic technique, the DHFR was the same for both the native and the trypsin- elution of the proteolyzed TS-DHFR was monitored by digested protein. DHFR activity assay and by NaDodSO4/PAGE. Fig. 1 shows the five individual 2-hr proteolytic digests, N112-Terminal Sequence Analysis. Sequential Edman degra- after NaDodSO4/PAGE. Each protease produced a fragment dation was performed by R. Harkins ofGenentech. An Applied of apparent Mr 35,000. V8 protease, trypsin, and a- Biosystems model 470A vapor-phase sequencer was equipped chymotrypsin also generated a polypeptide of Mr = 20,000. with a "mini" Conversion Flask and updated with the "no- When these various fragments were transferred to nitrocel- vacuum" program. Polybrene (1.5 mg) was used as a carrier in lulose and probed with antiserum raised against E. coli TS, the cup. In addition, the sequencer has been modified for only the Mr 56,000 subunit and the Mr 20,000 fragments automatic on-line HPLC separation of the phenylthiohydan- showed reactivity. When the fragments from a V8 digest were toin-amino acids as described by Rodriguez et al. (20). Se- probed with L. tropica TS-DHFR antibody, both the Mr quence data were interpreted with the aid ofa Nelson Analytical 36,600 and Mr 20,000 fragments as well as the Mr 56,000 model 6000 data-acquisition system, which was interfaced with subunit bound the antibody. Antiserum raised against E. coli the HPLC detector. The phenylthiohydantoin-amino acids DHFR showed no reactivity to the native TS-DHFR and was were resolved on a Microsorb C8 column (5-,um particle size, 4.6 not used in blot analysis. To test the assumption that all five mm x 25 cm). The column was operated at 42°C. The mobile Mr 35,000 fragments were formed by cleavage in a common phase consisted of 20o (vol/vol) CH3CN in 10 mM sodium region of the protein, these larger fragments were isolated phosphate (pH 4.5) (A) and 70%o CH3CN in water (B). The flow from gels and exposed to further proteolysis, with V8 rate was 1.5 ml/min and a gradient from 100o A to 100% B was protease (1:10 wt/wt) in 0.1% NaDodSO4. The resulting generated over 22 min. peptide maps were essentially the same (data not shown). Downloaded by guest on September 25, 2021 7190 Biochemistry: Garvey and Santi Proc. Natl. Acad. Sci. USA 82 (1985)

A 10 MTl 97- x 10 -'

68 - 9'7i - wt-0Mmw-"NW 68 - 43- 43 - * - ..T w =a3 an _ -'i -

- 18 -...... -t s is 0 10 2(0 30 40 85 120160220 18- Time (min)

1- B

I-2 it hn LI e f FIG. 1. NaDodSO4/PAGE analysis of limited proteolysis [2 hr, '4-4 25TC, 1:100 (wt/wt) ratio of protease to substrate] of L. tropica 0 to TS-DHFR by five different endopeptidases. Lane a: 5 Ag of intact C subunit. Lanes b-f: equivalent amounts of subunit, digested with V8 A4 protease, trypsin, a-chymotrypsin, elastase, or S. griseus protease, respectively. Proteins were visualized by staining with Coomassie blue R-250. Molecular weight standards: phospborylase b (97,000), bovine serum albumin (68,000), ovalbumin (43,000), a-chymotrypsin (25,000), 3-lactoglobulin (18,000), and cytochrome c (12,000). 160 Time (min) The time course of the V8 protease reaction with TS- DHFR is shown in Fig. 2. Initially, Mr 37,300 and Mr 20,000 FIG. 2. Time course of limited proteolysis (1:100, wt/wt) of L. fragments were produced; the larger fragment was further tropica TS-DHFR by the V8 protease. (A) Progression ofthe V8 proteolyzed, resulting in a more stable, Mr 36,600 fragment. protease reaction with TS-DHFR, as analyzed by NaDodSO4/PAGE At longer reaction times, the entire Mr 37,300 fragment was of aliquots removed at Indicated times. Molecular weight standards are as described in the legend to Fig. 1. (B) Time course of digestion converted to the Mr 36,600 fragment. Densitometric scanning as monitored by NaDodSO4/PAGE and DHFR (m) and TS (A) of gels from each time point showed a constant amount of activity. Stained bands representing the Mr 56,000 subunit (s) and Mr total protein, indicating that the Mr 36,000 and 20,000 37,300 (o), 36,700 (o), and 20,000 (A) fragments were quantitated by fragments were resistant to further proteolysis. After 220 scanning densitometry. min, -1Mo of the Mr 56,000 subunit remained, illustrating that both subunits of the native protein were cleaved during were identical to those shown in Fig. 2A, showing a lack of the reaction. a disulfide either between fragments or between subunits. To further characterize the fragments produced by V8 TS-DHFR was also treated with various exopeptidases. protease digestion, we attempted to separate the polypep- With a 1:10 (wt/wt) ratio of leucine aminopeptidase, amino- tides under nondenaturing conditions, using four techniques: peptidase M, or pyroglutamate aminopeptidase (alone or in nondenaturing gel electrophoresis, anion-exchange chroma- conjunction with aminopeptidase M), neither TS nor DHFR tography (DEAE-Sepharose), DHFR-specific affinity chro- activity was affected after 2 hr. In contrast, carboxypeptidase matography (methotrexate-Sepharose), and gel-permeation A (1:20, wt/wt) rapidly reduced TS activity in a time- chromatography (Sephadex G-150). With all four approach- dependent manner to 20% ofcontrol after 60 min; DHFR was es, the two fragments (which could be separated by NaDod- 90% of control at this time. Upon NaDodSO4/PAGE, no S04/PAGE) comigrated or were coeluted and behaved iden- difference in mobility of the Mr 56,000 subunit could be seen tically to the intact native dimer. With each approach, DHFR after any of the exopeptidase digestions. activity also comigrated or was coeluted with the two Previous attempts to determine the NH2-terminal sequence fragments. (i0 The proteolyzed TS-DHFR appeared as a of native TS-DHFR resulted in no derivatized amino acids single band upon nondenaturing PAGE; (it) the digested (5), suggesting that the NH2 terminus was blocked. From a protein bound to DEAE-Sepharose and was eluted at -75 NaDodSO4/polyacrylamide gel, we isolated the stable Mr mM KCl by use of a linear 0-200 mM gradient; (iii) the 36,600 and Mr 20,000 fragments produced by V8 protease and proteolyzed TS-DHFR adsorbed to methotrexate-Seph- subjected the peptides to automated Edman sequence anal- arose, was not removed by either high salt or mild detergents, ysis. The Mr 36,600 fragment failed to yield any sequence, and was competitively eluted with 7,8-dihydrofolate; and (iv) indicating that this fragment possessed the blocked NH2 the proteolyzed protein migrated with an apparent Mr 150,000 terminus of the native protein. Because the Mr 36,600 upon Sephadex G-150 chromatography, the same apparent fragment was derived from an initial Mr 37,300 fragment, the Mr as the native protein.t To determine whether a disulfide secondary cleavage by V8 protease must have occurred less bond held the fragments or the subunits together, the V8 than 10 residues from the COOH-terminal end of the Mr proteolysis and NaDodSO4/PAGE analysis were performed 37,300 fragment. In contrast to the blocked NH2 terminus of in the absence of thiols; the fragments observed on the gel the Mr 36,600 fragment, we obtained the sequence ofthe first 28 amino acids (with three omissions) of the Mr 20,000 3 shows this sequence with sequences tFrom amino acid analysis, the native TS-DHFR has a molecular fragment. Fig. aligned weight of 108,800. Upon gel filtration, the native protein has an from the four TSs that have been fully sequenced [human apparent molecular weight of 150,000 and a Stokes radius of 4.4 nm (21), E. coli (22), Lactobacillus casei (23), and T4 phage (24)]. (5). Each of these sequences occurs :'-20 kDa from the COOH- Downloaded by guest on September 25, 2021 Biochemistry: Garvey and Santi Proc. Natl. Acad. Sci. USA 82 (1985) 7191

L. tropica Met-Asp-Leu-Gly-Pro-Val-Tyr-Gly-Phe-Gin-Trp-Arg- and the Mr 36,600 fragment generated by V8 protease failed 129 to produce derivatized amino acids and the V8 protease- Human Gly-Asp-Leu-Gly-Pro-Val-Tyr-Gly-Phe-Gln-Trp-Arg- generated Mr 20,000 fragment yielded a free NH2 terminus, 88 we concluded that the Mr 36,600 fragment possesses the coli Gly-Asp-Leu-Gly-Pro-Val-Tyr-Gly-Lys-Gin-Trp-Arg- 140 blocked NH2 terminus of TS-DHFR and the Mr 20,000 L. casei Gly-Asp-Leu-Gly-Leu-Val-Tyr-Gly-Ser-Gfln-Trp-Arg- fragment represents the COOH terminus of the protein. 103 Gly-Glu-Leu-Gly-Pro-I le-Tyr-Giy-Lys-Gin-Trp-Arg- Third, when TS-DHFR was digested with either elastase or T4 phage the S. griseus type XIV protease, only the larger, Mr 35,000 fragments were stable, during which time the DHFR activity L. tropica Gly-Phe- X -Ala-Asp-Tyr-Lys- X -Phe-Glu-Ala-Asn- was unaffected and the TS activity was completely lost. More 141 extensive proteolysis of the Mr 35,000 fragments showed that Human His-Phe-Gly-Ala-Glu-Tyr-Arg-Asp-Met-Glu-Ser -Asp- these larger fragments were essentially the same. The larger 100 Thr- fragments, and therefore the NH2-terminal end of TS-DHFR, coli Aia-Trp-Pro 152 possess DHFR. Finally, there is a large degree of homology L. casei Ala-Trp-Hs .Thr- between the NH2-terminal sequence of the Mr 20,000 frag- 115 ment and sequences found ==20 kDa from the COOH-terminal T4 phage Asp-Phe-Gly end of TS from four sources. Also, only the Mr 20,000 fragments hybridized with an E. coli TS antibody. Therefore, part of TS resides on the Mr 20,000 fragment and, conse- L. tropica Tyr- X -Gly-Glu 153 quently, at the COOH-terminal end of TS-DHFR. Human Tyr-Ser-Gly-Gln-Gly-Val-Asp-Gln-Leu- Some of the above data also verify that the two subunits of 104 L. tropica TS-DHFR, which have the same size and charge E. coli Pro-Asp-Gly-Arg-His-Iie-Asp-Gln-Ile- (5), are indeed identical. Selective proteolysis of nonidentical 156 L. casei Ser-Lys-Gly-Asp-Thr-Ile-Asp-Gln-Leu- subunits is unlikely to occur at the same position in each polypeptide, as occurs in the V8 protease reaction. More T4 phage - - - - Gly-Vai-Asp-Gln-Ile- emphatically, nonidentical subunits are extremely unlikely to FIG. 3. Comparison of the NH2-terminal sequence at the Mr have an identical 28 amino acid internal sequence at the same 20,000 fragment from L. tropica TS-DHFR, generated by the V8 position of the polypeptide. The bifunctional TS-DHFR is protease, and homologous sequences found within TS from human found in a wide variety of protozoa. These proteins are (21), E. coli (22), L. casei (23), and T4 phage (24). The number above dimers of subunits of identical size (3-5), and it is likely that the first amino acid indicates the position of that amino acid in the the subunits are also identical. complete primary sequence. X indicates that the amino acid at that The model presented above of a DHFR sequence followed position was not identified. Dashes indicate gaps inserted to maxi- by a TS sequence, with at least some homology between the mize homology. The alignment of the human, E. coli, L. casei, and TS and non-protozoan TS, supports the suggestion sequences are Takeishi et al. (21). L. tropica T4 phage taken from that the L. tropica TS-DHFR gene (and most likely the terminal end ofthe protein. When the first 12 amino acids are TS-DHFR genes from other protozoa) resulted from the compared, 11 ofthe 12 positions are conserved in at least four fusion of independent TS and DHFR genes. In E. coli, the of the five synthases and 7 out of the 12 positions are genes encoding TS and DHFR map far apart on the chromo- conserved in all five synthases. The gaps within the E. coli, some (26, 27), but recent reports have shown a close L. casei, and T4 phage sequences shown in Fig. 3 were arrangement ofthe two separate genes in Bacillus subtilis (28) suggested by Takeishi et al. (21) and reflect the alignment and T4 phage (24, 29). Interestingly, in T4 phage, the codon necessary for continued homology among the reported se- for the COOH terminus of DHFR overlaps the codon for the quences of the four synthases. NH2 terminus of TS by a single base pair (24, 29), resulting in the biosynthesis of separate proteins. The introduction of DISCUSSION a single nucleotide into this overlap region found in T4 phage The bifunctional TS-DHFR from L. tropica is a dimer of could lead to synthesis of a bifunctional TS-DHFR. subunits with identical size (Mr 56,000) and charge (5). Our Many studies that have used limited proteolysis to study initial attempts to selectively cleave TS-DHFR and generate multifunctional proteins have revealed a susceptible hinge two stable fragments that reflected the size of non-protozoan region between catalytic or ligand-binding domains (6-9). TS and DHFR appeared to be successful. Not only did five The cleavage of such hinge regions has often allowed the different endopeptidases generate a stable fragment of M r isolation of functionally active domains (9). Although our 35,000 (Mr of non-protozoan TS is :35,000), but V8 protease data show that TS-DHFR consists of a DHFR sequence also produced a stable Mr 20,000 fragment (Mr of non- followed by a TS sequence, we have been unable to separate protozoan DHFR is -20,000). As data accumulated, how- distinct enzymatic domains by limited proteolysis. Rather, ever, we realized that our initial assignment of TS to the Mr the data indicate that upon digestion the subunits are initially 35,000 fragments and DHFR to the Mr 20,000 fragment severed within the TS domain of the protein. 'Tith V8 should in fact be reversed, and we concluded that the various protease, a second cleavage is subsequently made about 10 proteases did not make a scission directly between domains. residues toward the NH2-terminal end of the protein relative Data from the limited proteolysis of TS-DHFR indicate to the first cleavage. Under the conditions used, no other that DHFR and TS sequences are arranged in a linear fashion proteolysis by V8 protease was detected. Surprisingly, these on the polypeptide, with DHFR at the blocked NH2-terminal cleavages do not disrupt the gross, overall integrity of the and TS at the COOH-terminal end of the protein. First, TS, subunits. Four different separation techniques under but not DHFR, activity is lost upon digestion with carboxy- nondenaturing conditions failed to resolve fragments; gel- peptidase A. This inactivation of TS results from the hydrol- permeation chromatography, in particular, demonstrated ysis of only a few COOH-terminal amino acids, since the that the proteolyzed subunits do not dissociate. Proteolysis migrations of the carboxypeptidase A-treated and native and analysis of proteolytic products in the absence of a proteins could not be differentiated upon NaDodSO4/PAGE. reducing agent indicated that neither the fragments nor the [A similar inactivation of TS from L. casei by carboxypep- subunits were held together by a disulfide bond. tidase A has been reported (25).] Second, because attempts In contrast to this apparent lack of effect on higher-order to sequence the NH2 terminus of both the native TS-DHFR structure, kinetic data show that proteolysis does disrupt Downloaded by guest on September 25, 2021 7192 Biochemistry: Garvey and Santi Proc. Natl. Acad Sci. USA 82 (1985) some subunit structure and subunit-subunit interactions. Not affecting overall subunit stability. It is not known whether only is TS activity rapidly lost upon proteolysis, it is lost these various insertions represent functional peptide, but we approximately twice as fast as the subunit is cleaved, so that suggest two possibilities. First, these insertions might fold when TS activity is completely lost, -50% of the subunits into a functional higher-order structure and enable TS to bind remain uncleaved. One reasonable explanation is that within a multienzyme complex; TS activity has been reported proteolysis of one subunit causes perturbations within that to be associated with such a complex in T4 phage-infected subunit and also between subunits, inactivating TS in both; bacteria (30) and in mammalian cells (31). Second, TS activity hence, the 2-fold difference in rates. Curiously, the disturb- is exceedingly labile during preparation of crude extracts ance of quaternary structure does not affect the ability of TS from L. tropica when protease inhibitors were omitted (5). to bind its stoichiometric inhibitor FdUMP in the presence of This important metabolic enzyme activity may be regulated cofactor; that is, TS loses this ability at the same rate as V8 in vivo through proteolysis, with these insertions providing a protease cleaves TS-DHFR and at about one-half the rate of target for endopeptidases. TS inactivation. A second possible effect of proteolysis on quaternary structure is the increase of methotrexate bound We thank Ric Harkins of Genentech for performing the NH2- after digestion. Previously, we showed that only one mole of terminal sequence analysis. This work was supported by Public the DHFR inhibitor methotrexate is bound per mole of Health Service grant 19358. D.V.S. is a Burroughs Wellcome Scholar TS-DHFR dimer (5). As shown here, the amount of in Molecular Parasitology. methotrexate bound per mole of TS-DHFR dimer increased 1. Blakely, R. L. (1984) in Folates and Pteridines, eds. Blakely, R. L. 2-fold after proteolysis by trypsin. Proteolysis appears to & Benkovic, S. J. (Wiley, New York), Vol. 1, pp. 191-253. disrupt the negative cooperativity observed between 2. Santi, D. V. & Danenberg, P. V. (1984) in Folates and Pteridines, subunits in the intact dimer and to free a second methotrex- eds. Blakely, R. L. & Benkovic, S. J. (Wiley, New York), Vol. 1, ate-binding site. pp. 343-396. A large degree of homology exists among the complete 3. Garrett, C. E., Coderre, J. A., Meek, T. D., Garvey, E. P., Cla- (21), E. man, D. M., Beverley, S. M & Santi, D. V. (1984) Mol. Biochem. primary structures of TS from human coli (22), L. Parasitol. 11, 257-265. casei (23), and T4 phage (24); 45-60o homology is found 4. Ferone, R. & Roland, S. (1980) Proc. Natl. Acad. Sci. USA 77, when any two sequences are compared. The 28 amino acid 5802-5806. internal sequence of TS-DHFR aligns with internal se- 5. Meek, T. D., Garvey, E. P. & Santi, D. V. (1985) Biochemistry 24, quences found at approximately the same position in the four 678-686. TS sequences. The first 12 positions of these five sequences 6. Mattick, J. S., Tsukamoto, Y., Nickless, J. & Wakil, S. J. (1983) J. Biol. Chem. 258, 15291-15299. show striking homology; 7 of the 12 positions are identical 7. Walker, M. S. & DeMoss, J. A. (1983) J. Biol. Chem. 258, and only 1 position shows even moderate variability. The 3571-3575. TS-DHFR sequence shows the highest degree of homology 8. Matthews, R. G., Vanoni, M. A., Hainfeld, J. F. & Wall, J. (1984) when compared with human TS: 11 of the first 12 positions J. Biol. Chem. 259, 11647-11650. are identical, 17 of the total 28 positions are identical, and, 9. Grayson, D. R. & Evans, D. R. (1983) J. Biol. Chem. 258, where differences occur, only a few are significant. When the 4123-4129. 10. Hillcoat, B., Nixon, P. & Blakely, R. L. (1967) Anal. Biochem. 21, last 16 positions ofthe 28 amino acid sequence are compared, 178-189. the L. tropica sequence only shows homology with human 11. Wahba, A. J. & Friedkin, M. (1961) J. Biol. Chem. 236, PC11-12. TS. Both sequences appear to possess insertions in this 12. Santi, D. V., McHenry, C. S. & Sommer, H. (1974) Biochemistry region relative to the sequences of the three prokaryotic 13, 471-481. synthases. The human TS sequence possesses two consec- 13. Laemmli, U. K. (1970) Nature (London) 227, 680-685. utive 14. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-421. glutamate residuesjust prior to the sequence that aligns 15. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. with the internal 28 amino acid sequence of TS-DHFR; the Sci. USA 76, 4350-4354. specificity of V8 protease (which generated the M, 20,000 16. Fisher, P. A., Berrios, M. & Blobel, G. (1982) J. Cell Biol. 92, fragment) is for peptide bonds involving acidic amino acids. 674-686. Therefore, it is likely that the L. tropica sequence also shows 17. Blake, M. S., Johnston, K. H., Russell-Jones, G. J. & Gotschlich, a glutamate at this position. E. C. (1984) Anal. Biochem. 136, 175-179. 18. Fischer, S. G. (1983) Methods Enzymol. 100, 424-430. Insertions of polypeptide are found within the TS primary 19. Hunkapiller, M. W., Lujan, E., Ostrander, F. & Hood, L. E. structures when the four sequences are compared. One ofthe (1983) Methods Enzymol. 91, 227-236. most striking examples is found relative to the E. coli 20. Rodriguez, H., Kohr, W. J. & Harkins, R. N. (1984) Anal. sequence. A 51 amino acid insertion between amino acids 87 Biochem. 140, 538-547. and 88 of E. coli occurs within the L. casei TS (20). A 13 21. Takeishi, K., Kaneda, S., Ayusawa, D., Shimizu, K., Gotoh, O. & amino acid insertion is found at the same point within the Seno, T. (1985) Nucleic Acids Res. 13, 2035-2043. human TS sequence. Finally, a 9 amino acid insertion is 22. Belfort, M., Maley, G., Pedersen-Lane, J. & Maley, F. (1983) Proc. found within the T4 phage sequence. The NH2 terminus ofthe Natl. Acad. Sci. USA 80, 4914-4918. 23. Maley, G. F., Bellisario, R. L., Guarino, D. U. & Maley, F. (1979) Mr 20,000 fragment generated by the V8 protease coincides J. Biol. Chem. 254, 1301-1304. with position 88 of the E. coli TS and precisely with the 24. Chu, F. K., Maley, G. F., Maley, F. & Belfort, M. (1984) Proc. COOH-terminal end of the insertions found in L. casei, Natl. Acad. Sci. USA 81, 3049-3053. human, and T4 phage TS. In addition, the molecular weight 25. Aull, J. L., Loeble, R. B. & Dunlap, R. B. (1974) J. Biol. Chem. of each TS, measured from these positions to the COOH 249, 1167-1172. terminus, is =20,000. Because the five proteases used in this 26. Taylor, A. L. & Trotter, C. D. (1967) Bacteriol. Rev. 31, 332-353. study initially cleave TS-DHFR in the same region (V8 27. Breeze, A. S., Sims, P. & Stacey, K. A. (1975) Genet. Res. 25, protease cleaves a second time in this area), this region most 207-214. likely is exposed on of the 28. Myoda, T. T., Lowther, S. V., Fananage, V. L. & Young, F. E. the surface protein. Upon (1984) Gene 29, 139-147. cleavage, TS-DHFR remains a dimer and does not dissociate 29. Purohit, S. & Mathews, C. K. (1984) J. Biol. Chem. 259, into fragments, indicating that the bulk of inter- and 6261-6266. intrasubunit forces are unaffected by proteolysis of this 30. Allen, J. R., Reddy, G. P. V., Lasser, G. W. & Mathews, C. K. region. Given this model, an insertion is also likely to occur (1980) J. Biol. Chem. 255, 7583-7588. within the L. tropica TS, positioned at the surface of the 31. Reddy, G. P. V. & Pardee, A. B. (1980) Proc. Natl. Acad. Sci. protein, vulnerable to the action of endopeptidases, but not USA 77, 3312-3316. 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