Vol. 265, No. 21, Issue of July 25, pp. 12546-12552, 1990 Printed in U.S.A.

Clp P Represents a Unique Family of Serine Proteases*

(Received for publication, March 1, 1990)

Michael R. MauriziS, William P. Clark, Seung-Ho Kim& and Susan Gottesman From the Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892

The amino acid sequence of Clp P, the proteolytic subunit of the ATP-dependent Clp protease of Esche- richia coli, closely resembles a protein encoded by chloroplast DNA, which is well conserved between chloroplasts of different plant species. The homology extends over almost the full length of the sequences of both proteins and consists of -46% identical and -70% similar amino acids. Antibodies against E. coli Clp P cross-reacted with proteins with M, of 20,000-30,000 in bacteria, lower eukaryotes, plants, and animal cells. Since the regulatory subunit of Clp protease, Clp A, also has a homolog in plants, as well as in other bacteria and in lower eukaryotes, it is likely that ATP-depend- ent proteolysis in chloroplasts is catalyzed in part by a Clp-like protease and that both components of Clp-like proteases are widespread in living cells. We have iden- tified Ser- 111 as the active site serine in E. coli Clp P modified by diisopropyl fluorophosphate. Mutational alteration of Ser- 111 or His- 136 eliminates proteolytic activity of Clp P. Both residues are found in highly conserved regions of the protein. The sequences around the active site residues suggest that Clp P represents a unique class of serine protease. Amino-terminal proc- essing of cloned Clp P mutated at either Ser-111 or His-136 occurs efficiently when wild-type clpP is pres- ent in the chromosome but is blocked in clpP- hosts. Processing of Clp P appears, therefore, to involve an intermolecular autocatalytic cleavage reaction. Since processing of Clp P occurs in clpA- cells, the autopro- cessing activity of Clp P is independent of Clp A.

In the previous paper (l), we described the cloning, map- ping, and sequencing of the gene for the ClpP subunit of the ATP-dependent Clp protease of Escherichia coli. Cleavage of proteins such as casein and albumin by Clp protease requires both Clp P and the regulatory subunit, Clp A and the contin- uous hydrolysis of ATP (2-5). However, Clp P alone was shown to have limited peptidase activity against a fluorogenic dipeptide in the absence of Clp A (6), and recent experiments’ indicate that Clp P alone can catalyze endoproteolytic cleav- age of short peptides, such as oxidized insulin, at about l-2% of the rate observed in the presence of Clp A and ATP. Thus, Clp P appears to possess within its primary structure the essential elements needed to catalyze peptide bond cleavage.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: National Cancer Institute, Bldg. 37, Rm. 2E20, Bethesda, MD 20892. Tel.: 301-496- 7961; Fax: 301-496-0450.

§ Current address: Genetic Engineering Center, Korea Advanced Institute of Science and Technology, P. 0. Box 131, Cheongryang, Seoul, Korea.

’ M. R. Maurizi, manuscript in preparation.

Hwang et al. (4) reported that Clp P was inactivated by diisopropyl fluorophosphate, suggesting that Clp P is a serine protease. The open reading frame for Clp P does not contain any sequences similar to those for trypsin-like or subtilisin- like proteases, suggesting that Clp P may represent a unique class of serine protease. The very low intrinsic activity of Clp P and the need for tight binding of Clp A to activate prote- olysis by Clp P raise the question of whether or not Clp P has the aspartate and histidine that along with serine make up the catalytic triad found at the active site of most serine proteases.

Recently, ClpA has been shown to be a member of a highly conserved family of polypeptides, found in a variety of pro- karyotic and eukaryotic organisms (7). E. coli itself contains at least one other member of this family, called cl@. The degree of overall conservation in these proteins (>50% simi- larity) and the extraordinary conservation in >150-amino acid regions including the ATP-binding pockets (>70% similarity) strongly suggested that these proteins may be part of Clp-like energy-dependent proteases in the various organisms. This conclusion would be strengthened if the proteolytic subunit, Clp P, were also found to be conserved in the same cells that have the Clp A homolog.

In this paper, we report that Clp P is present in a wide range of prokaryotic and eukaryotic cells and that Clp P is in fact highly conserved in plant chloroplasts, suggesting that ATP-dependent Clp-like proteases are present in plants and possibly all organisms. We also show that Clp P of E. coli has a serine and a histidine that are necessary for activity and which probably represent two elements of the active site triad found in most serine proteases.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids-The E. coli strains used were derivatives of MC4100 (8). SG21164 has an NruI-EcoRV deletion of the promoter and 5’-coding region of +A (3), AclpA164, and SG22007 has the deletion/insertion clpPl::CM (1). Plasmids pWPC9 (cl@+) and pWPCl0 (cZpPM14L) were described in the accompanying paper (I). Plasmids pWPCl1 (clpPS111A) and pWPC12 (clpPHl36Q) are described below. Strain RZ1032 dut ung is described by Kunkel (9).

Assays of C&J Protease and Clp P Activity-Clp protease was assayed by the release of acid-soluble products from radioactively labeled casein as described previously (2). For assay of Clp P in various strains, 5-ml cultures in Luria broth (with 100 Kg/ml ampi- cillin when appropriate) were grown to Aa” 2 1.0 and harvested by centrifugation. The cells were frozen before suspending in 0.5-1.0 ml buffer B (2) without dithiothreitol, containing 1 mg/ml lysozyme. After 15-30 min on ice, the cells were sonicated in 5-s pulses for 20 s, and cell debris was removed by centrifugation for 5 min in an Eppendorf microcentrifuge. Clp P activity in cell extracts was deter- mined as the difference in ATP-dependent casein degradation when assays were performed with and without addition of excess purified Clp A.

Purification of Clp A and Clp P-Clp A was purified from an overproducing strain as described previously (3). Clp P purification is described in the accompanying paper (1). Mutated Clp P was partially purified by heating clarified extracts at 60 “C for 10 min,

12546

Clp-like Serine l’rotease Family

followed by gel filtration on a Bio-Rad TSK250 column (0.75 X 40 cm) in buffer B (1, 2).

Preparation of Affinity Purified Anti-Up P Antibody-CNBr-acti- vated Sepharose was washed with 1 mM HCl and then water, before mixing with an equal volume of HEPES* buffer, pH 8.0, containing 2 mg/ml purified Clp P. After shaking for 1 h at room temperature and 3 h at 4 OC, the gel was washed in a funnel with HEPES buffer to remove unreacted Clp P. The gel was incubated for 3 h with 1 M glycine, titrated to pH 9.0 with KOH, washed with buffer B without dithiothreitol, and stored at 4 ‘C. The gel contained approximately 2 mg/ml Clp P covalently attached. Antibody was prepared by passing 0.5 ml of serum (containing 1 M KC1 and 1% Nonidet P-40) three times over a small column of Clp P-Sepharose (0.2 ml). The column was washed with 3-4 ml of 20 mM Tris/HCl, pH 7.5, containing 1 M NaCl and 1% Nonidet P-40 and then with 1 ml of 20 mM Tris buffer containing 150 mM NaCl. Antibody was eluted with 8 M urea in the same buffer and diluted by dropwise addition into 20 volumes of 20 mM Tris, pH 7.5, with 150 mM NaCl, 0.05% Tween 20, and 5% non- fat dried milk.

Isolation of the Clp P Peptide Modified by DFP-Purified Clp P (400 gg) was incubated in buffer B (without dithiothreitol) with 10 mM [3H]DFP (10 &i/pmol) for 3 h at room temperature. Radioactive Clp P was precipitated with 67% acetone, dissolved in buffer B with dithiothreitol, and precipitated a second time with 67% acetone. One- half of the sample was treated with 0.1 M CNBr in 70% formic acid for 3 h at room temperature (10). Reagents were removed by lyophi- lization, and the CNBr peptides were dissolved in 1% SDS with 1% /3-mercaptoethanol and loaded on a Vydac Cl8 reverse-phase column (0.75 x 25 cm). Peptides were eluted with a linear gradient of O-60% acetonitrile and were detected at 210 nm (11). Aliquots of the peptide fractions were counted and the sample containing most of the radio- activity was dried in a Speed-Vat concentrator (Savant).

Amino Acid Analysis-Hydrolysis of peptides was carried out at 155 ‘C in 6 N HCl for 40 min. HCl was removed under vacuum, and the amino acids were dissolved in water. Aliquots were mixed with an equal volume of o-phthalaldehyde reagent, which was composed of 0.5 M sodium borate, pH 10.5, 20 mM o-phthalaldehyde, 70 mM fl- mercaptoethanol, and 1.5% methanol. After 30 s the sample was injected onto an IBM Cis reverse-phase column (4.5 X 150 mm) and eluted at 30 ‘C with a gradient of methanol and 0.075 M NaCl (11).

Mutagenesis of the Cloned clpP-The Ser-111 to Ala and the His- 136 to Gin mutations in Clp P were made by oligonucleotide-directed mutagenesis in uitro essentially as described by Kunkel (9). The clpP+-Ml3 mpl8 DNA was isolated from phage grown in strain RZ1032 and was replicated in uitro using synthetic oligonucleotides as primers. The oligonucleotide primer used to replace the serine was complementary to nucleotides 694-723 in Fig. 6 of the accompanying paper (l), except that the A complementary to T-709 was changed to C. Similarly, the histidine replacement was accomplished with an oligonucleotide primer complementary to nucleotides 771-800, except that the G complementary to C-785 was changed to a C. The in uitro replicated phage were plated on a lawn of strain DH5a from Bethesda Research Laboratories, plaques were grown up, and phage DNA was isolated. The presence of the insert was confIrmed by restriction enzyme analysis, and the incorporation of the base substitution in the DNA was determined by sequencing. The mutated clpP genes were transferred to pBR322 by digestion of the phage DNA with BarnHI and Hind111 and ligation with pBR322 DNA cut with the same enzymes (1).

Other Procedures-Methods for protein determination, SDS-gel electrophoresis, Western blotting, etc. were described in the accom- panying paper or references therein (1).

RESULTS

Consermtion of Clp P-The open reading frame coding for the Clp P protease bears no strong resemblance to either the other well-studied energy-dependent protease of E. cob, Lon, or to other previously sequenced proteases. A search of the NBRF protein sequence data bank (12) for open reading frames with homology to Clp P showed an extremely similar protein coded for by the chloroplast DNA of tobacco and

* The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid DFP, diisopropyl fluorophosphate; SDS, sodium dodecyl sulfate; ORF, open reading frame; HPLC, high per- formance liquid chromatography.

liverwort (Fig. 1). A clpP-like gene is also found in the rice chloroplast genome recently sequenced by Hiratsuka et ul. (13). The predicted proteins are all of similar length: 207, 196, 203, and 217 amino acids for the E. cob, tobacco, liverwort, and rice proteins, respectively. The amino-terminal 14 amino acids of the open reading frame for E. coli Clp P, which are not found in the mature protein (1), are not homologous to the chloroplast sequences. The amino acid sequences of the mature E. coli Clp P and the chloroplast homologs can be aligned along their entire lengths with only a single one- amino acid gap. Pairwise comparisons of sequences are shown in Table I. Clp P shares 36-46% identical and 60-70% similar plus identical amino acids with the different chloroplast pro- teins, with liverwort showing the greatest, and rice the least, similarity to E. coli Clp P. There are 31% identical and 53% similar plus identical amino acids shared by all four proteins. The degree of conservation between E. coli Clp P and the chloroplast Clp P homologs is comparable to that observed between E. coli and tobacco chloroplast ribosomal proteins,

51 . l . . . . A t A . . . *a * . .A* . . * * . ” “ ” . l A . . . * . ” . “,-lrJr)

Nodi ClpP cc?rmNLIvAfl l4LrLNANNPN I tD IYL~INSP GGvITAaMsI YlmMQrmPD

lbbacco ClpP sErsN*LIGL MvYLsmDE* KDLYmINsP GGwvIPGvN rml4QwRPD

Lit=awort ClpF DEnN*LIGI lwn.lmmES K O M Y L Y I N S P GGA~GISV YDAMWWPD

Ric. ap* cEvmmITGL MvYLs~EDos SDmLnNSP GGNLXSQIN FLnnQTvTPN

101,. ***..* ** . * . . “ A * A . . “ A . . * t . . . “ A ” . . - . * * A 150

E C O U c&@ VsTxzMGQAA LLmG zaKGKRFcLPN 0. YQWATDI

lk.bacco fxp VNTICMG~ IL”GG E I -PIi S~XEAQTGEW

r.iv.rlm* C1F.F VEITCMGLM rn?rGG *rrFaIALPN ! S * Y D G Q A G N C

Rice ClpP IxlTCLGmA lTIJ&G EmFammN HYRARTPEF

151.. “ A . A ..a . , . . . . . “ ” A , . ” l ** .-a * . * ““A”2OlJ

NCOli apP EInAFxnxv KM- mGQsLE*IN P.DTENom.¶ A P E A V E Y G L ”

Tobacco ap* VLEAEErn ru$TLTrnQ RTGKeLNvvS Em- *mAQAYG~”

Li~wcl@O~ C1p.P nLEAENvLKL RDc1mvYvQ RTmmvIs Em- AIomKLYGI”

Rice up* LLNvEELNF.v REMImvmL r?lsKems E D - ALCSkKAWX.”

*01* . 226

Neali ClpP D .$ ILTNPN. . . . . . . . . . . . . . . . . . . .

mk.*cco c1p* r n , v A v E . . . . . , , . . . . . . . . . . . . . . .

Li”erwo* ClpP o~~~-~~*~.......,.......

Rice ClpP DIvGDEkaDE NCrJTDwhTP ~KDW

FIG. 1. Comparison of the amino acid sequences of E. coZi Clp P and three chloroplast open reading frames. The E. coli Clp P sequence was obtained in this laboratory (see accompanying paper, Ref. 1). The chloroplast sequences for liverwort (Murchantia polymorphu, ORF203 (31)) and tobacco (Nicotiunu tobucum, ORF 196 (32)) were retrieved from the NBRF protein sequence data bank (12). The sequence for rice (Oryza satioa) chloroplast Clp P (ORF216) was from Hiratsuka et ul. (13). The positions at which the reading frames are interrupted by introns in the liverwort and tobacco genes are indicated by the oerticul urrows. The serine and histidine residues necessary for Clp P activity are boxed (see later experiments for details). Identical amino acids occurring in the same position of all four proteins are indicated by an asterisk; similar amino acids in the same position of all four proteins are indicated by a (-).

TABLE I Amino ucid conservation between E. coli Clp P

und chloroplust homologs The numbers above the diagonal are the percent identical amino

acids occurring in the same positions in the proteins. Numbers below the diagonal refer to the percent identical plus similar amino acids in the corresponding positions in the proteins. Similarity was based on the Dayhoff evolutionary comparisons (30). Sequences of Clp P from amino acid 15 to the end and the overlapping portions of the chloro- plast sequences were compared. Tbe Clp P-like protein sequences were translated from chloroplast DNA sequences from liverwort (M. polymorpha) (31), tobacco (N. tobacam) (32), and rice (0. satiuu) (13).

E. co& Liverwort Tobacco Rice E. coli 46.2 42.4 36.2 Liverwort 69.5 78.5 60.7 Tobacco 67.0 so.3 68.9 Rice 60.2 82.1 85.2

12548

which have 36-68% similarity (14).

Clp-like Serine Protease Family

The tobacco and liverwort chloroplast genes contain introns that interrupt the reading frame after amino acids 60 and 132 (indicated by a uertical arrow in Fig. 1). The reading frame in exon 2 is more conserved among the four proteins (43% identical and 65% similar plus identical) than those in exon 1 (18% identical, 33% similar plus identical) or exon 3 (27% identical, 50% similar plus identical). As described below, exon 2 contains the active site serine.

TABLE II

Inactivation of Ck P with DFP

Addition” Activity remaining

Clp P Trypsin

None %

100 100

The hydropathy profiles (15) of Clp P and the chloroplast homologs are very similar, except for the less-well conserved regions between amino acids 15 and 35 and amino acids l75- 183 in Clp P, which have a more hydrophilic character in the E. coli protein (data not shown). Secondary structure predic- tions (16) throughout most of the four proteins are also very similar (data not shown). The most significant differences occur between amino acids 150 and 180 of Clp P, which is predicted to form two a-helices in Clp P and two p-sheets in the chloroplast homologs. This region of the sequences is poorly conserved between Clp P and the chloroplasts proteins, although it is highly conserved among the chloroplast proteins themselves. Whether these differences in predicted structures actually exist in the proteins must be determined experimen- tally, but it seems probable that this region of the protein serves different structural or functional roles in the E. coli and the chloroplast Clp P proteins.

0.2 mM DFP 100 0 2 mM DFP 89 0 2 mM DFP + Clp A/MgATP 90 ND 1OmM DFP - 3 ND

” Clp P or trypsin was incubated at room temperature in buffer B without dithiothreitol with DFP for 30 min and then diluted into a standard casein degradation assay mixture. DFP was added in a final concentration of 10% isopropanol which had no effect on either activity. The Clp A added (1 pg) was sufficient to saturate the Clp; the concentrations of Mg and ATP were 25 and 2 mM, respectively.

23,000 protein that reacts strongly with anti-Clp P antibody.” It seems highly likely that these 20-30-kDa proteins are homologs of Clp P. The 23-kDa cross-reactive protein from spinach (lane g) may be the Clp P homolog encoded in chloroplasts. The recent finding of Tanaka et al. (17) that anti-Clp P antibody reacts with components YCla and YClb of the yeast proteasome is consistent with the conclusion that the proteolytic function of Clp P has been conserved in at least some of these proteins. We do not know if the protein seen with whole yeast extract in Fig. 2 (Mr - 27,000) is the same as the proteasome components whose reported molecu- lar weight (-30,000) is somewhat higher.

Presence of Clp P-like Proteins in Other Organisms-The extensive homology between Clp P and the chloroplast pro- teins suggested the possibility that Clp P-like proteins in different organisms might possess antigenic determinants in common with Clp P. Proteins from various tissues and orga- nisms were screened by Western blotting for proteins that cross-reacted with anti-Clp P antibody. Fig. 2 shows that affinity purified anti-Clp P antibody reacted with proteins found in several bacteria (lanes h-k), eukaryotic microorga- nisms (lanes a-c), plants (lane g), and animal cell lines (lanes d-0. In addition, cross-reactive proteins were detected in a number of rat tissues (data not shown). These proteins were not detected when nonimmune rabbit serum was used (data not shown).

The bacteria, lower eukaryotes, and plants all have cross- reactive proteins with molecular weights between 21,000 and 30,000. In addition to these, Bacillus subtilis also has an Mr

ll6K 97 K

- 66 K

- 43 K

- 31 K w

m 0 4 CIPP

- 21 K

- -14K

FIG. 2. Western blot of various prokaryotic and eukaryotic cell extracts with antibody against E. coZi Clp P. About 50 pg 01 protein from each different organism or tissue was separated on 10% polyacrylamide gels in SDS. Western blots were developed with affinity-ljurified anti-Up P antibody as described previously (1). Lanes contain extracts from: a, yeast; b, Acanthamoeba, c, Dktyoste- llurn, d, Chinese hamster ovary cells; e, liver hepatoma cells; 1, NIH XT3 cells; g, spinach leaves; h, M. uannieli; i, Clostridium thermoace- t~urn, j, Ckxtridium kluyveri, h, Rickttsia. Lane l has purified Clp P.

Several higher molecular weight proteins also were found to cross-react with the anti-Clp P antibody. Acanthamoeba castellonii (lane b) and the archebacterium Methanococcus uannieli (lane h), both contain a z40-kDa cross-reactive pro- tein, and several transformed animal cell lines possess a very large cross-reactive protein (~80 kDa). Further experiments will be required to determine the extent of the structural similarity or conservation between Clp P and these proteins.

Identification of the Active Site Serine in Clp P-Hwang et

al. (4) had shown that DFP inactivated proteolytic activity of Clp protease and covalently modified the Clp P subunit, suggesting that Clp P contained an activated serine residue characteristic of serine proteases. Table II shows that inacti- vation of Clp P with DFP required relatively high concentra- tions of DFP compared to those required to inactivate trypsin. Inactivation with phenylmethylsulfonyl fluoride was also very slow and incomplete (data not shown). Activation of Clp P for proteolysis by addition of Clp A and ATP did not increase the rate of reaction with DFP (Table II). No sequences found around the active site serine, histidine, or aspartate residues in either trypsin-like or subtilisin-like serine proteases were present in the amino acid sequence of Clp P. Thus, the active site of Clp P might be expected to differ from other serine proteases, and it was important to establish that DFP was in fact reacting with a serine in the active site of Clp P.

Incubation of Clp P with [“H]DFP for 3 h at room temper- ature resulted in incorporation of 0.90 & 0.1 nmol of diisopro- pylphosphoryl adduct/nmol Clp P; Clp P incubated in parallel with nonradioactive DFP was completely inactivated (data not shown). To identify the active site serine in Clp P, the protein labelled with [“H]DFP was cleaved with cyanogen bromide, and CNBr fragments were separated by HPLC (Fig. 3). Essentially all of the [“H]DFP incorporated into Clp P was recovered in a single peptide. Amino acid analysis of the isolated peptide after hydrolysis in 6 N HCl gave 1 glycine, 1 glutamate, 1 serine, 2 alanines, and 1 homoserine. From the

,’ R. L. Switzer and L. Bussey, personal communication.

Clp-like Serine Protease Family 12549

genesis, the clpP gene was altered to replace this histidine with a glutamine. Extracts of transformants carrying this mutant plasmid, pWPCl2 (clpPHl36Q), contained expected amounts of the mutant Clp P protein, detected by Western blots (see Fig. 4 below). Clp P activity, however, was absent from these extracts (Table III), indicating that the Clp P- Hl36Q protein is inactive. This result suggests that His-136 is part of the active site triad of Clp P.

FIG. 3. Separation of cyanogen bromide fragments of Clp P labeled with [“H]DFP. Purified (Zip P (150 pg), radioactively la- beled with [“H]DFP was digested with CNBr and the resulting peptides were separated on a Vydac Cjti reverse-phase column. Pep- tides were detected by absorbance at 210 nm (.&id !kze) and collected as they were eluted from the column. Aliquots flO% of’ each fraction) were assayed for tritium by liquid scintillation counting (A).

TABLE III l’rotwl\f~c actfctt\ of wild-type and mutated C’lp I’

None Clp I’* CLP P-Ml4L Clp P-SlllA 0.16 O.l<i Cm P-HlXQ 0.14 0.11

” Assays were carried out as described previously (2) using -10 pg of protein from clarified cell extracts. The host cells were SC22007 fc/pf’J::CMl. The mcrease in ATP-dependent casein degradation when excess Clp A is added to the assay mixtures is a measure of Clp P activtty.

sequence of Clp P, only one peptide flanked by methionines could give rise to this composition, and the active site serine was identified as Ser-111 (boxed in Fig. 1).

To confirm the involvement of this serine in nroteolvsis. we used oligonucleotide-directed mutagenesis. with d the Ml3mpl8-clpP+ clone to change the codon for Ser-111 of Clp P to that for an alanine (see “Experimental Procedures”). The mutant gene, clpPSlllA, was cloned into pBR322, and transformants with the c@PSlllA plasmid were shown to overproduce the mutant Clp P protein detected by Western blots with anti-Clp P antibody (see Fig. 4, below). Clp P assays using extracts of clpP1::CM host cells transformed withpclpPSlllA revealed no detectable Clp P activity (Table III), indicating that the Clp P produced from the mutant plasmid was inactive. Therefore, Ser-111 of Clp P is essential for Clp protease activity and is most likely the active site serine for this ATP-dependent protease. The sequence around Ser-111 is highly conserved in the chloroplast Clp P homologs (Fig. 1). A search of both NBRF sequences and translated GenBank sequences with the boxed sequence around the active site serine did not reveal any proteins other than those from chloroplasts with this unique sequence.

Processing of Clp P in clpP Mutants-In the preceding paper (1) it was shown that Clp P is synthesized with a 14- amino acid precursor peptide at the amino terminus. Removal of this peptide to generate the native 193-amino acid Clp P is very rapid. A mutant (clpPMl4L) in which the methionine at the site of cleavage was replaced by a leucine was also rapidly processed, apparently at the same site (1). As seen in Fig. 4, only the fully processed form of Clp P was found in ion- (lane c) and clpA- cells (lane cl), indicating that neither Lon protease nor Clp A is required for processing of Clp P. Overproduction (>20-fold) of Clp P from a plasmid did not appear to saturate the processing system, since essentially all the Clp P produced from a wild-type clpP plasmid was processed. No significant amounts of the unprocessed form accumulated in either clpP+ or clpP- host cells (Fig. 4, lanes f and k) or in clpA- cells (data not shown). Also, when the plasmid carried clpPMl4L, proc- essing of the Clp P was complete in both wild-type and mutant host cells (lanes g and l). In contrast, cells carrying plasmids with mutated clpP (clpPSlllA or clpPHl36Q), which synthe- size inactive Clp P, had significant amounts of unprocessed Clp P protein. In clpP+ hosts, 30-50X of the inactive Clp P protein was processed (Fig. 4, lanes h and i). When these clpP mutant plasmids were present in a clpP:CM mutant host, the plasmid-encoded Clp P was totally unprocessed (Fig. 4, lanes m and n). This requirement for an active clpP either in the chromosome or on the plasmid to obtain processing of Clp P suggests that the active protease itself is needed for cleavage of its own precursor. The processing of the plasmid-encoded mutant protein in the presence of wild-type chromosomally encoded Clp P demonstrates that the cleavage can occur in an intermolecular reaction. This cleavage of its own precursor peptide is the first detection in uiuo of an activity for Clp P in the absence of Clp A.

Gel Filtration of Unprocessed Clp P-Extracts of clpP- cells over-producing Clp P-Hl36Q from pWPCl2 were heated at 60 ‘C and applied to an HPLC gel filtration column in buffer B plus 0.2 M KCl. Wild-type and mutant Clp P protein were recovered in about the same amounts. Clp P was detected in the fractions by dot-blot analysis with anti-Clp P antibody.

FIG. 4. Western blots of Clp P made from multicopy clpP- plasmids. Cells bearing either wild-type or mutant c!pJl plasmids were grown to mid-exponential phase and the cultures were quenched with trichloroacetic acid. Protein from each culture was separated on 10% gels and analyzed by Western blotting with anti-Clp P antibody. Lanes b, c, d, e, and j were loaded with 100 pg of protein from nontransformed cells; all other lanes contained 25 pg of protein from different transformants. Host strains carried either c/pf>+ Clatws b-i) or c/p1’1::CM (/anesj-n) in the chromosome. Lane, a, purified Clp P; /am b, wild-type cells: lane c, /on- cells; [ane d, C/PA- cells: /ants 1 and k, pWPC9 (+I’+); /arm ,g and /, pWPCl0 fc/pJ’Ml4Ll; /arm /r and m, pWPCl1 fclpK3lllA); [arws i and n, pWPCl2 fc/p1>HlXQ).

His- 136 Is Necessary for Clp P Activity-The active sites of most previously described serine proteases use the side chains of a histidine and an aspartate together with the serine to form the catalytic center for proteolysis. Comparison of the sequences of Clp P with those of the ClpP-like proteins from chloroplasts revealed 6 aspartates but only 1 histidine con- served in all four proteins. The conserved His-136 lies within a stretch of seven amino acids perfectly conserved in E. coli, tobacco, and liverwort. Using oligonucleotide-directed muta-

Clp-like Serine Protease Family

Unprocessed Clp P emerged as a slightly higher molecular during incubation in vitro under the above conditions (data weight protein (Mr 280,000) than processed Clp P (M, not shown), this result indicates that the amino-terminal 240,000) (data not shown). Thus, both processed and unproc- extension on the Clp P precursor does not prevent binding of essed Clp P appear to have the same quaternary structure Clp P to Clp A. We can not at present determine whether the and exhibit similar heat stabilities, indicating that removal of extension would interfere with activation of Clp P by Clp A, the precursor peptide of Clp P is not required for folding or because the unprocessed form of Clp P with a wild-type active self-association of Clp P. site is not available.

Clp P Mutated at the Actiue Site Binds to Clp A-In uitro experiments with Clp P inactivated with DFP indicated that the modified protein could bind to Clp A. Fig. 5 shows that the proteolytic activity of Clp P in the presence of limiting Clp A was inhibited by increasing amounts of DFP-inacti- vated Clp P. Inhibition was about 50% when the active and inactive proteins were present in equal amounts, When Clp A was in sufficient excess to saturate both active Clp P and inactive Clp P, no inhibition was observed. This result shows that DFP-modified Clp P competes with active Clp P for binding to and activation by Clp A.

DISCUSSION

To determine whether the mutations of the active site serine or histidine produced major conformational changes in Clp P, the ability of the mutated Clp P proteins to compete with active Clp P for binding to Clp A was tested. Table IV shows that mutated Clp P from transformants carrying plas- mids pWPCl1 (clpPSlllA) or pWPCl2 (clpPHl36Q) added to assay solutions with limiting Clp A inhibited casein deg- radation by Clp P. No inhibition was observed if Clp A was in excess (data not shown). Thus, in the presence of limiting Clp A, mutant Clp P and active Clp P appear to compete for binding to the Clp A. No significant differences in the amounts of inactive Clp P needed for inhibition were seen whether the Clp P was totally unprocessed (from clpP1::CM host) or partially processed (from clpP+ host). Since Western blots showed that the unprocessed Clp P was not cleaved

The earlier finding that Clp P was covalently modified and inactivated by DFP suggested that Clp P was a serine protease (4). In this paper we have identified the serine modified by DFP (Ser-111) and shown that mutating that serine elimi- nated activity of Clp P. Mutational alteration of His-136 to a glutamine also eliminated enzymatic activity. While the loss of activity in the His-136 mutant does not, by itself, prove that His-136 is the active site histidine, two other findings support this conclusion. First, His-136 is the only conserved histidine between the E. coli and the chloroplast proteins. Second, the mutant Clp P proteins appear to have the same native size as active Clp P, they are processed similarly, they have similar heat stability, and the mutant proteins can interact efficiently with Clp A, indicating that the histidine to glutamine mutation did not cause gross conformational changes in Clp P.

The positions of the active site serine and histidine in the primary structure of Clp P and the sequences around those residues are well conserved in the Clp-like proteins of chlo- roplasts, but these sequences differ from those found in other serine proteases. The amino acid composition around the active site serine most resembles that for subtilisin-like serine proteases which contain glycine, methionine, and alanine close to the serine (Table V), but the sequence of the amino acids is different. The active site residues for the ATP- dependent Lon protease have not been identified, but Chin et ul. (18) reported that none of the serines in Lon protease are found in a sequence context similar to that found in the trypsin or subtilisin families of serine proteases. We have found no sequence similarities between Lon protease and Clp protease around the active site serine. Sequences around His- 136 also bear no resemblance to those found in other serine proteases or in Lon protease. The position of the histidine on the carboxyl-terminal side of the active site serine is more reminiscent of sulfhydryl proteases, such as papain, than of serine proteases, in which the order of occurrence of these residues in the peptide chain is usually aspartate-histidine- serine. The aspartate residue that is part of the active site triad of classical serine proteases has not yet been identified in Clp P. There are five aspartates that are conserved in all four proteins, but none of them is surrounded by sequences found around known active site aspartates. Thus, while at least two of the basic components of the serine protease catalytic mechanism seem to have been conserved in Clp protease, the differences in specific sequences around the

-0 2 4 6 6 10

[DIP-Clp P]/[Clp P]

FIG. 5. Competition between active and inactive Clp P for binding to Clp A. Active Clp P (1 fig) was mixed with different amounts (O-8 fig) DFP-inactivated Clp P and assayed for casein degradation in the presence of limiting Clp A (A) or with excess Clp A WI.

TABLE IV

Mutant Clp P added to inhibit Cm P+ Clp P activity’

A. Pure Clp P (no extracts) B. None

Clp P-SlllA (2:l) Clp P-SlllA precursor (2:l) Clp P-Hl36Q (2:l) Clp P-Hl36Q precursor (2:l)

j.~g ca.sein/h %

4.6 100 4.6 100 1.5 32 1.5 32 0.73 16 0.88 19

’ Standard casein degradation assays were carried out with limiting amounts of Cln A and excess Clp P (either pure Clp P in part A or from extracts of a transformant &ith the Clp P+ plasmid in part B). Extracts of transformants containing mutant Clp P were added in 2- fold excess over the wild-type extracts to measure competition be- tween the forms of Clp P.

TABLE V Sequemes around active site serines in serine proteases

Sequences were obtained from the NBRF protein sequence data bank. The putative active site serine of Lex A was identified by Slilaty and Little (20). The serine in Umu D was identified by Nohmi et ul. w.

Clp P GQAASMGA Chloroplasts GLAASMGS LexA VSGMSMKD Umu D ASGDSMID Subtilisin DGTSMAS Trypsin-like GDSGGP

Clp-like Serine Protease Family

important residues suggest that Clp represents a unique fam- ily of serine proteases.

The rather weak reactivity of Clp P with DFP and phenyl- methylsulfonyl fluoride indicates that the active site serine is not activated to the same degree in Clp P as in the trypsin or subtilisin families of serine proteases or that the serine is not as accessible to these reagents. Activation of Clp P by Clp A apparently does not involve a simple opening up of the pro- teolytic active site of Clp P, since Clp A does not increase the reactivity of Clp P with DFP. Inactivation of Lon protease (18) and of Lex A4 also requires relatively high concentrations of DFP, suggesting that the active site serines of ATP-de- pendent proteases in general are weak nucleophiles, which may in part account for their rather low turnover numbers (11, 19). The Lex A (20) and Umu D (21, 22) proteins, which have limited auto-degradative activities, possess a serine in the proteolytic active site (Table V) which does not appear to be activated by the classical charge-transfer mechanism in- volving histidine and aspartate. These proteins are activated for self-cleavage by interaction with Ret A (23, 21), which may lead to a conformational change positioning a lysine residue to activate the reactive serine (20).

mains and a mechanism for the preferential presentation of the substrate portions of the conjugates into the proteolytic active sites. The mechanism by which Clp protease transfers bound substrates to the active site could be somewhat analo- gous to that used by the 26 S protease, but clearly Clp protease must possess a different mechanism for the recognition of appropriate substrates. In the ubiquitin-dependent degrada- tion system, recognition of appropriate protein targets for degradation is the function of the E3 components of the conjugating system (28), whereas Clp protease (and Lon pro- tease) apparently contains the recognition function within the protease itself. Elucidation of this function in Clp protease will perhaps help remove the major stumbling block to our understanding the control of intracellular protein breakdown in all cells.

A&rzo&edgment-We would like to thank K. Rudd for assistance in retrieving the sequence for the rice chloroplast ORF 216.

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