©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Potential Active Site of the Signal Peptidase SipS of Bacillus subtilis
STRUCTURAL AND FUNCTIONAL SIMILARITIES WITH LexA-LIKE PROTEASES (*)

(Received for publication, September 16, 1994; and in revised form, December 5, 1994)

Jan Maarten van Dijl(§)(¶) Anne de Jong (¶) Gerard Venema Sierd Bron (¶)

From the Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN, Haren (Gn), The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Signal peptidases remove signal peptides from secretory proteins. By comparing the type I signal peptidase, SipS, of Bacillus subtilis with signal peptidases from prokaryotes, mitochondria, and the endoplasmic reticular membrane, patterns of conserved amino acids were discovered. The conserved residues of SipS were altered by site-directed mutagenesis. Replacement of methionine 44 by alanine yielded an enzyme with increased activity. Two residues (aspartic acid 146 and arginine 84) appeared to be conformational determinants; three other residues (serine 43, lysine 83, and aspartic acid 153) were critical for activity. Comparison of SipS with other proteases requiring serine, lysine, or aspartic acid residues in catalysis revealed sequence similarity between the region of SipS around serine 43 and lysine 83 and the active-site region of LexA-like proteases. Furthermore, self-cleavage sites of LexA-like proteases closely resembled signal peptidase cleavage sites. Together with the finding that serine and lysine residues are critical for activity of the signal peptidase of Escherichia coli (Tschantz, W. R., Sung, M., Delgado-Partin, V. M., and Dalbey, R. E. (1993) J. Biol. Chem. 268, 27349-27354), our data indicate that type I signal peptidases and LexA-like proteases are structurally and functionally related serine proteases. A model envisaging a catalytic serine-lysine dyad in prokaryotic type I signal peptidases is proposed to accommodate our observations.


INTRODUCTION

The most important common feature of protein transport across the cytoplasmic membrane of prokaryotes and the endoplasmic reticular (ER) (^1)membrane of eukaryotes is the fact that transported proteins are synthesized with an amino-terminal signal peptide. The signal peptide is required for protein targeting to the membrane and initiation of protein translocation across the membrane (1, 2, 3, 4) . Although the primary structure of signal peptides is poorly conserved, three domains can be distinguished in all of them: first, a short amino-terminal domain with at least one positively charged amino acid; second, a central hydrophobic domain (h-region) of 7-15 amino acids; and third, a polar carboxyl-terminal domain (c-region) of 3 to 7 amino acids(3) . Structurally related signal peptides are required for protein transport to the mitochondrial intermembrane space(5) . During translocation of the protein across the membrane, the signal peptide is removed (processing) by signal peptidases (SPases; (6) ). The c-region specifies the SPase cleavage site(7, 8) . Turn-promoting residues that are carboxyl-terminal to the cleavage site are required for efficient processing(9) .

The prokaryotic SPases, which recognize typical signal peptides as described above, are known as type I SPases, or leader peptidases (Lep). These membrane proteins process the majority of exported bacterial pre-proteins(10, 11) . Three type I SPases of Gram-negative bacteria have been characterized. These are Lep (EC) of Escherichia coli(10) , Lep (ST) of Salmonella typhimurium(12) , and Lep (PF) of Pseudomonas fluorescens(13) . Lep (ST) and Lep (PF) show a high degree of sequence identity with Lep (EC) (93 and 50%, respectively) over almost their entire length. As demonstrated for Lep (EC), these three proteins possess two amino-terminal membrane anchors, and both their amino- and carboxyl-terminal moieties are exposed to the periplasm(14) .

Recently, we identified SipS of Bacillus subtilis, which is the first characterized type I SPase of a Gram-positive bacterium(15) . Although SipS shows some sequence similarity with Lep (EC/ST/PF), it is structurally quite distinct from the latter enzymes(15, 16) . First, SipS (184 residues) is much smaller than Lep (EC/ST/PF) (323, 324, and 284 residues, respectively). Second, three regions of Lep (EC/ST/PF) are absent from SipS. One of the missing regions corresponds to the first membrane anchor of Lep (EC/ST/PF). Unexpectedly, SipS showed a much higher degree of similarity with a subunit (Imp1p) of the mitochondrial inner membrane protease I (ImpI) of Saccharomyces cerevisiae(17) , which processes proteins targeted to the inter-membrane space. Like SipS, Imp1p has only one membrane anchor, and both proteins share similarities over almost their entire length (27% identity; (15) ). The alignment of SipS, Lep (EC/ST), and Imp1p revealed patterns of conserved amino acids in five distinct regions (denoted A-E) of each enzyme (Fig. 1). These patterns were also detectable in the Imp2p subunit of ImpI(18, 19) , the Sec11 subunit of the ER SPase complex (SPC) of S. cerevisiae(20) , and the two subunits Spc18 and Spc21 of the canine ER SPC(21, 22) . Thus, with the sequence of SipS, we could demonstrate structural similarities between prokaryotic and eukaryotic SPases, indicating that these proteins belong to one class of enzymes(15) .


Figure 1: Patterns of conserved amino acids in prokaryotic and eukaryotic SPases. The comparison includes the deduced amino acid sequences of SipS of B. subtilis(15) , Lep (EC) of E. coli(10) , Lep (ST) of S. typhimurium(12) , Lep (PF) of P. fluorescens(13) , the Imp1p and Imp2p subunits of the mitochondrial inner membrane protease I of S. cerevisiae(17, 18) , the Sec11 subunit of the ER SPC of S. cerevisiae(20) , and the Spc18 and Spc21 subunits of the canine ER SPC(21, 22) . Five subregions, A-E, containing the most conserved patterns of amino acids, are shown. To facilitate the comparison, the SPases were divided into three groups: first, prokaryotic SPases (SipS, Lep (EC), Lep (ST), Lep (PF)); second, the mitochondrial Imp1p and Imp2p; and third, eukaryotic SPases (Sec11, Spc18, Spc21). Identical residues or conservative changes are only boxed when present in at least two of the groups of SPases. Consequently, in some cases, nonconserved amino acids are present in one box. Numbers refer to the position of the first amino acid of each subregion in the respective SPases. Residues important for the activity (*) and conformation (up triangle) of SipS are indicated.



In the present study we investigated which residues are required for SipS to be functional. We concentrated on those residues that are conserved in all type I SPases because we anticipated that these would be the most important. The residues of region A (Fig. 1) were not analyzed because they reside in the membrane anchor of SipS. This part of the protein is probably not required for the catalytic reaction, because a truncated soluble form of Lep (EC), lacking both anchors, is still capable of correct signal peptide cleavage(23) .


MATERIALS AND METHODS

Plasmids, Bacterial Strains, and Media

Table 1lists the plasmids and bacterial strains used. TY medium (tryptone/yeast extract) contained Bacto-tryptone (1%), Bacto-yeast extract (0.5%), and NaCl (1%). M9 medium-1 and -2 used in the labeling of E. coli were prepared as described in (24) . Both M9 media were supplemented with ampicillin (40 µg/ml).



DNA Techniques

Procedures for DNA purification, restriction, ligation, and agarose gel electrophoresis were carried out as described in (25) . Enzymes were from Boehringer (Mannheim, Germany). E. coli MC1061 was electrotransformed with ligation mixtures, using a Bio-Rad Gene Pulser. Polymerase chain reactions (PCR) were carried out with the Vent DNA polymerase (New England Biolabs). For site-directed mutagenesis of SipS residues in region B, the following protocol was used. Chromosomal template DNA of B. subtilis was denatured for 1 min at 94 °C. Next, two primers (primer M and primer B; Table 2were used to amplify DNA fragments in 20 cycles of denaturation (30 s; 94 °C), primer annealing (1 min; 50 °C), and DNA synthesis (1.5 min; 73 °C). In addition to the desired mutations, primer M specified a SalI site. Primer B specified either a BclI or an EcoRI site (Table 2). Amplified fragments were purified with the Qiagen PCR purification spin kit (Qiagen Inc., Chatsworth, CA), digested overnight with appropriate restriction enzymes, and ligated to linearized plasmid DNA (pMO). When a mutation was to be introduced in the middle part of a DNA fragment (e.g. mutations in regions C, D, and E of SipS), two subsequent PCR reactions were performed. A first PCR with a mutagenic primer (primer M; Table 2) and limiting amounts of a primer specifying one end point of the desired fragment (primer B; Table 2) was performed as described above. Next, about 0.2 µg of the amplified fragment from the first PCR was used as a primer in the second PCR step together with a third primer (primer A; Table 2) specifying the other end point of the desired fragment. This second PCR was carried out under the same conditions as the first PCR. The sequences of all DNA fragments obtained through PCR were verified by the dideoxy-chain termination method(26) , using the T7 sequencing kit (Pharmacia, Uppsala, Sweden). S-dATP was from Amersham (Radiochemical Centre, Amersham, UK). DNA and protein sequences were analyzed using version 6.7 of the PCGene Analysis Program (Intelligenetics Inc., Mountain View, CA). The FASTA algorithm (27) was used for protein comparisons in the Swiss Protein Data base (release 18), the Atlas of protein and genomic sequences (release September 1993; MIPS, Martinsried, FRG), and the EMBL Nucleotide Sequence Data Bank. The RDF2 program was used to evaluate sequence similarities(28) . To calculate z values, the KTUP value was set to 2, and 500 random shuffles of the test sequences were performed. Alignments with z values greater than 6 were considered significant; alignments with z values smaller than 3 were considered insignificant.



Pulse-Chase Protein Labeling, Immunoprecipitation, SDS-PAGE, and Fluorograph

Pulse-chase labeling, immunoprecipitation, and SDS-PAGE were performed as described in Refs. 15, 24, and 29. ^14C-Methylated molecular weight markers were from Amersham International. Fluorography was performed as described in (30) . Relative amounts of precursor and mature forms of (A13i)-beta-lactamase were estimated by film scanning with an LKB ultroscan XL laser densitometer. The activities of SipS mutants were compared with that of wild-type SipS by comparing the relative amounts of mature (A13i)-beta-lactamase obtained after a chase period of 10 min or 3 h in the presence of the wild-type or the mutant enzyme.

SipS antiserum

A SipS-based peptide (sippep 1) with the sequence NKKRAKQD (residues 115-122 of SipS) was synthesized by Eurosequence B.V. (Groningen, The Netherlands). Sippep 1 contained an amino-terminal S-acetylmercaptoacetyl group to facilitate its cross-linking to Imject^R maleimide-activated bovine serum albumin from Pierce(31) . Rabbits were used to raise antibodies against the peptide-bovine serum albumin conjugate at the Finnish National Public Health Institute (KTH; Helsinki).

Western Blot Analysis

The presence of SipS in E. coli C600 and B. subtilis 8G5 sipS was assayed by Western blotting(32) . Lysates of E. coli C600 were prepared by concentrating cells from overnight cultures 7-fold in loading buffer for SDS-PAGE and subsequent boiling (5 min). Membranes of B. subtilis 8G5 sipS were prepared from cultures in TY medium(33) . Membrane proteins were solubilized in 2% Triton X-100 (30 min, 0 °C). Nonsolubilized material was removed by centrifugation (135,000 times g; 20 min; 4 °C). After SDS-PAGE, proteins of E. coli C600 or solubilized membrane proteins of B. subtilis 8G5 sipS were transferred to Immobilon-polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Bands corresponding to SipS were visualized with antibodies against sippep 1 and alkaline phosphatase-anti-rabbit-IgG conjugates (Western-light, Tropix, Bedford, MA).

Prediction of SPase Cleavage Sites

SPase cleavage sites were predicted according to the algorithms of von Heijne(34) . The same algorithms were used in a computer program, enabling the search for SPase cleavage sites with a variable window. Sequences of 15 amino acids (residues -13 to +2) that yield a ``von Heijne score'' higher than 3.5 strongly resemble SPase cleavage sites. For shorter sequences (e.g. -5 to +2) von Heijne scores greater than 0 indicate a strong resemblance with regions surrounding SPase cleavage sites.


RESULTS

Mapping of Functionally Important Conserved Residues of SipS

To map functionally important conserved residues of SipS, a site-directed mutagenesis approach was used. Residues of SipS, which are conserved in all type I SPases, were replaced by alanine. Alanine was chosen because it is small, and it has a chemically inert side chain. Thus, conformational strain and indirect effects on catalysis were minimized. Mutations were introduced in the 3` part of sipS (encoding residues 41-184) by PCR, using the oligonucleotides shown in Table 2. These were designed in such a way that amplified fragments contained a SalI site at the 5` end and an EcoRI or a BclI site at the 3` end (Table 2). After restriction with SalI and EcoRI, or SalI and BclI, the fragments were ligated to plasmid pMO (Fig. 2), which was cut with the same restriction enzymes. Since pMO contains the 5` end of the sipS gene (encoding the residues 1-40), this resulted in a complete copy of a mutant sipS gene. Another advantage of pMO was that it encodes the hybrid precursor pre(A13i)-beta-lactamase, which is processed by SipS but not by Lep (EC)(15, 24) . Thus, pre(A13i)-beta-lactamase could be used directly to monitor the activity of SipS mutants. To this purpose, E. coli C600 cells containing mutant sipS genes (encoded by pMO derivatives) were labeled for 1 min with [S]methionine and, subsequently, chased with an excess of nonradioactive methionine for 10 min. With wild-type SipS (E. coli C600 (pGDL41)), this resulted in approximately equal amounts of precursor and mature forms of labeled (A13i)-beta-lactamase (Fig. 3, lane +). From a total of 30 SipS mutants in which a conserved residue had been changed to alanine, 11 showed approximately wild-type activity (Fig. 3; T47A, R68A, V72A, V79A, G87A, L88A, P89A, G90A, D91A, Y141A, and N150A). Six SipS mutants showed a slightly reduced activity, ranging between 57 and 90% of the wild-type activity (Fig. 3; P46A, G69A, D70A, I71A, I86A, and S154A). Six other mutants (L48A, L74A, Y81A, N147A, S151A, and R155A) had a more strongly reduced activity ranging between 5 and 39% of the wild-type activity (Fig. 3; L74A and R155A; for other mutants, see Fig. 4). Finally, six mutants (S43A, K83A, R84A, G145A, D146A, and D153A) appeared to be inactive (not shown in Fig. 3; see Fig. 4). To verify the (in-)activity of SipS mutants of the latter two categories (strongly reduced or no activity), pulse-chase labeling experiments were performed in which the chase period was extended to 3 h (Fig. 4). Also in these experiments, the SipS mutants S43A, K83A, and R84A were completely inactive. The mutants D146A and D153A had almost completely lost activity; at the most 3-3.5% of the total (A13i)-beta-lactamase synthesized was mature. In contrast, the activities of the mutants L48A, Y81A, G145, N147A, and S151A were clearly manifest after 3 h of chase (Fig. 4). These data indicated that serine 43, lysine 83, arginine 84, aspartic acid 146, and aspartic acid 153 were the most important (conserved) residues for the functionality of SipS.


Figure 2: Schematic presentation of pMO. Only restriction sites relevant for the construction and properties of the plasmids are shown. pMO contains the 5` end of sipS (nucleotides 1-352). The 3` end of sipS (nucleotides 353-796) was replaced by the sequence (5`-3`) GTCGACGGGAATTCCCTGATCA, specifying a multiple cloning site (SalI-EcoRI-BclI). Thus, pMO encodes the first 40 residues of SipS. Mutant sipS genes (*) with site-specific alterations in the sequences that specify the conserved regions B, C, D, or E (indicated with b, c, d, or e) were obtained by introducing PCR-derived SalI-EcoRI or SalI-BclI fragments (encoding residues 41-184 of SipS) in pMO. bla, 5`-truncated TEM-beta-lactamase gene; the wild-type signal sequence was replaced by signal sequence A13i(15) ; Km, kanamycin resistance marker; Em', disrupted erythromycin resistance marker.




Figure 3: Pre(A13i)-beta-lactamase processing by SipS mutants. Processing of pre(A13i)-beta-lactamase in E. coli C600 was analyzed by pulse-chase labeling at 37 °C and subsequent immunoprecipitation, SDS-PAGE, and fluorography. Cells were labeled with [S]methionine for 60 s prior to chase with excess nonradioactive methionine. Samples were withdrawn 10 min after the chase. The SipS mutants analyzed are indicated. Variations in the amounts of label in different lanes relate only to variability in the incorporation of label into cells of different cultures and not to specific effects of certain SipS mutants. p, precursor; m, mature; R, molecular mass marker (30 kDa); + or -, pre(A13i)-beta-lactamase processing, respectively, in the presence (E. coli (pGDL41)) or absence of wild-type SipS (E. coli (pGDL42)).




Figure 4: Pulse-chase analysis of pre(A13i)-beta-lactamase processing by SipS mutants with severely reduced activity. Processing of pre(A13i)-beta-lactamase was analyzed as in Fig. 3, but samples were withdrawn 3 h after the chase. The SipS mutants analyzed are indicated. p, precursor; m, mature; R, molecular mass reference (30 kDa); +, processing of pre(A13i)-beta-lactamase in the presence of wild-type SipS (E. coli C600 (pGDL41)); -, processing of pre(A13i)-beta-lactamase in the absence of SipS (E. coli C600 (pGDL42)).



Interestingly, one SipS mutant (M44A) showed a 1.4-fold increased activity as compared with the wild type (Fig. 3). To analyze the role of methionine 44 in more detail, pulse-chase experiments were performed. In the presence of mutant M44A, the half-life of pre(A13i)-beta-lactamase was approximately 3 min, as compared with 10 min in the presence of wild-type SipS (not shown). This observation indicated that methionine 44 is important for the level of SipS activity.

Serine 43, Lysine 83, and Aspartic Acid 153 Are Critical for the Activity of SipS, and Arginine 84 and Aspartic Acid 146 Are Important Conformational Determinants of SipS

To evaluate the importance of the residues of SipS that could not be replaced by alanine without a severe loss of activity, these residues were also replaced by other, if possible, more related residues. Thus, serine 43 was replaced by cysteine, threonine, and valine. Interestingly, serine 43 could be replaced by cysteine and threonine without complete loss of activity. Nevertheless, the activity of the mutants S43C and S43T was highly reduced compared with that of the wild type (Fig. 4); only 15 and 34%, respectively, of all (A13i)-beta-lactamase synthesized was processed after 3 h of chase. The mutant S43V showed no activity at all (Fig. 4). Thus, serine 43 appeared to be essential for optimal activity of SipS.

To assess the possible role of lysine 83 and arginine 84, these residues were replaced by histidine. In addition, lysine 83 was replaced by arginine and arginine 84 by lysine. Also, a double mutant K83R,R84K was constructed. Lysine 83 appeared to be critical for activity of SipS because it could not be replaced by histidine or arginine without complete loss of activity (Fig. 4). In contrast, the replacement of arginine 84 by lysine (Fig. 3) did not affect the activity of SipS. When arginine 84 was replaced by histidine, the activity of SipS was strongly reduced (Fig. 4); only 10% of all (A13i)-beta-lactamase was processed after 3 h of chase. These data indicated that arginine 84 is less important for SipS activity than lysine 83. Finally, the double mutant K83R,R84K was inactive (Fig. 4), which is in line with the observation that lysine 83 could not be replaced by arginine.

To investigate the importance of the aspartic acid residues at positions 146 and 153, these residues were also replaced by glutamic acid or asparagine. Aspartic acid 146 could be replaced by glutamic acid without loss of activity (Fig. 3). In contrast, the activity of the mutant D146N was severely reduced (Fig. 4); after 3 h of chase only 8% of all (A13i)-beta-lactamase was mature. Changes of aspartic acid 153 to glutamic acid or asparagine resulted in a nearly complete loss of SipS activity (Fig. 4); after 3 h of chase only 1-2% of all (A13i)-beta-lactamase was processed in the presence of these mutants, indicating that aspartic acid 153 is very important for SipS to function.

Since an increased protease sensitivity might cause the reduced activity of SipS mutants, the presence of several mutant proteins in E. coli C600 was tested by Western blotting (Fig. 5A). To probe further the protease sensitivity of the SipS mutants, they were introduced in B. subtilis, an organism which is known for its high-level production of extracellular proteases(35) . Subsequently, the presence of the mutant proteins in B. subtilis was verified by Western blotting (Fig. 5B). For this purpose, we used the B. subtilis strain 8G5 sipS, which lacks the chromosomal sipS gene. None of the mutations at position 43 (S43A, S43C, S43T, and S43V) affected the stability of SipS in E. coli or in B. subtilis. The same was true for SipS mutants in which lysine 83 or aspartic acid 153 was replaced by other residues (Fig. 5, A and B). Together with the finding that replacement of serine 43, lysine 83, and aspartic acid 153 by other residues (almost) completely abolished the activity of SipS, these data indicated that serine 43, lysine 83, and aspartic acid 153 are critical for SipS to function.


Figure 5: Identification of SipS mutant proteins with severely reduced activity. The integrity of SipS mutant proteins in E. coli C600 (A), and B. subtilis 8G5 sipS (B) was tested by SDS-PAGE and Western blotting. Lysates of E. coli C600 cells and solubilized membrane proteins of B. subtilis 8G5 sipS were prepared as described under ``Materials and Methods.'' The SipS mutant proteins analyzed are indicated. WT, wild-type SipS (A, E. coli C600 (pGDL41); B, B. subtilis 8G5 sipS (pGDL41)); -, no SipS (A, E. coli C600 (pMO); B, B. subtilis 8G5 sipS (pMO)). Arrows indicate the position of SipS.



Two SipS mutants (D146A and D146N) were unstable in E. coli and B. subtilis (Fig. 5, A and B). In contrast, the SipS mutant D146E was stable in E. coli (Fig. 5A) but not in B. subtilis (Fig. 5B). Similarly, the SipS mutants R84A and R84H were stable in E. coli (Fig. 5A) and unstable in B. subtilis (Fig. 5B). The only mutant that was unstable in E. coli and stable in B. subtilis was the double mutant K83R,R84K (Fig. 5, A and B). These data suggest that the inactivity of the SipS mutants R84A, R84H, and K83R,R84K and the low activity of the SipS mutants D146A and D146N relate to an altered conformation, rendering these mutant proteins sensitive to protease. Thus, arginine 84 and aspartic acid 146 seem to be important conformational determinants of SipS.

Aspartic Acid 91 Is Not Important for the Activity of SipS

Although aspartic acid 91 of SipS could be replaced by alanine without loss of activity (Fig. 3), the importance of this residue was further investigated because the corresponding aspartic acid residue of Lep (EC) (aspartic acid 153; Fig. 1) could not be replaced by alanine without loss of activity(36) . Aspartic acid 91 of SipS was replaced with glutamic acid or asparagine. Both mutants D91E and D91N showed wild-type activity (Fig. 3). These observations indicate that aspartic acid 91 is not important for the activity of SipS.

Nonconserved Residues at Positions 40, 41, and 42 Are Important for Activity of SipS

Although the residues between the conserved regions A and B of SipS and Lep (EC) are relatively well conserved (15) , the three residues immediately preceding serine 43 of SipS and serine 90 of Lep (EC) are not conserved. Since serine 43 appears to be critical for SipS activity, we investigated whether these three nonconserved residues are important for the functionality of SipS. The role of glycine 41 of SipS was of particular interest since this residue is only conserved in Imp1p and Imp2p. In all other type I SPases a glycine residue resides immediately amino-terminally of the serine residue, corresponding to serine 43 of SipS (Fig. 1). First, aspartic acid 42 of SipS was changed to serine. This had no serious effect on the SipS activity (Fig. 3), indicating that aspartic acid 42 is not critical for function. Subsequently, two double mutants of SipS were constructed in which glycine was placed amino-terminally of serine 43. First, glycine 41 and aspartic acid 42 were swapped. The resulting SipS mutant G41D-D42G was not active (Fig. 4). This was not due to degradation of the protein, because its presence could be demonstrated by Western blotting (Fig. 5, A and B). A second double mutant was constructed in which glycine 41 and aspartic acid 42 were replaced by serine and glycine, respectively (like in Lep (EC)). This SipS mutant (G41S,D42G) was still active. However, its activity was low (Fig. 4); only 15% of all (A13i)-beta-lactamase was mature after 3 h of chase. Finally, to determine whether the activity of the SipS mutant G41S,D42G might improve upon the introduction of a proline residue at position 40 (like in Lep (EC); (15) ), the triple mutant D40P,G41S,D42G was constructed. This mutant was, however, completely inactive (Fig. 4), which may be due to an altered protein conformation; although the SipS mutant D40P, G41S,D,42G was stable in E. coli (Fig. 5A), it was unstable in B. subtilis (Fig. 5B). These data indicated that the order of the three residues at positions 40, 41, and 42 is important for SipS to function.

SipS Is Structurally and Functionally Related to LexA and LexA-like Proteases

So far, the involvement of serine and lysine residues in catalysis has been demonstrated only for two homologous proteolytic enzymes of E. coli, which perform self-cleavage reactions. These are LexA and UmuD(37) . The active site residues of these enzymes (serine 119 and lysine 156 of LexA; serine 60 and lysine 97 of UmuD) are conserved in a large group of LexA-like proteases (Fig. 6A). To determine whether these proteases are structurally related to SipS, their amino acid sequences were compared with that of SipS. Interestingly, the region which is most conserved in all LexA-like proteases showed a high degree of sequence similarity with the region of SipS between valine 39 and isoleucine 86 (Fig. 6A). SipS showed the highest degree of similarity with SamA and ImpA of S. typhimurium. Furthermore, SipS showed a high degree of similarity with UmuD, MucA, DinR, and the LexA proteins of E. coli, S. typhimurium, and Erwinia carotovora. A lower degree of sequence similarity was found with other homologues of LexA, such as the CI repressor of bacteriophage and the repressor of bacteriophage P22 (not shown). Most importantly, serine 43 and lysine 83 of SipS aligned with serine 119 and lysine 156 of LexA (EC) and the conserved serine and lysine residues in the other LexA-like proteases. In addition, glycine 41 and arginine 84, which are important for the activity of SipS, are conserved in the LexA-like proteases (Fig. 6A). Since most residues of SipS, which are conserved in LexA, are also conserved in type I SPases (Fig. 6A, regions B, C, and D), these data could mean that type I SPases and LexA-like proteases are mechanistically related enzymes. Therefore, we investigated whether the self-cleavage sites of the LexA-like proteases resemble type I SPase cleavage sites. Indeed, with the algorithms of von Heijne(34) , we could show that all self-cleavage sites of LexA-like proteases resembled the SPase cleavage sites (c-regions) of signal peptides, eukaryotic signal peptides in particular (Fig. 6B). In most LexA-like proteases, the identification of (putative) self-cleavage sites required the use of search windows comprising little more than the c-region. However, the self-cleavage site of UmuD, showing the highest similarity to SPase cleavage sites, could be predicted with a search window including part of the h-region (Fig. 6B). Finally, we investigated whether mutations that reduce the efficiency of self-cleavage of LexA (38) and CI (39) would reduce the probability of these sites as putative SPase cleavage sites. Indeed, several mutations at the positions -5 (G80V), -3 (V82E, V82M), and -1 (A84D, A84T, A84Delta) reduced the SPase cleavage probability of the self-cleavage site of LexA (not shown). This was also true for a mutation (A111T) at the position -1 of the self-cleavage site of CI. Other self-cleavage site mutations in CI, replacing a glycine residue at the +1 position (G112A, G112E, G112R), severely affected the turn probability of this region (not shown). Thus, similar to SPase cleavage sites, turn-promoting residues seem to be required for efficient self-cleavage. Taken together, our observations indicate that SipS and the LexA-like proteases are mechanistically related enzymes, recognizing similar substrates.


Figure 6: Similarities between LexA-like proteases and SipS. A, patterns of conserved amino acids in LexA-like proteases and SipS. The comparison includes, with the EMBL/GenBank/DDBJ accession numbers in parentheses: LexA (EC) of E. coli (P03033); LexA (ST) of S. typhimurium (P29831); LexA (ER) of E. carotovora (X63189); DinR of B. subtilis (P31080); MucA of S. typhimurium (P07376); UmuD of E. coli (P04153); SamA of S. typhimurium (P23831); ImpA of S. typhimurium (P18641); and SipS (Z11847). The percentages of identical and similar residues in each LexA-like protease and SipS are shown. z values indicate the significance of alignments with SipS (28) (see ``Materials and Methods''). The conserved regions B, C, and D of SipS are indicated. Residues critical for the activity of LexA (EC) and SipS are shown (*). B, self-cleavage sites of LexA-like proteases. Putative type I SPase cleavage sites were identified in LexA (EC/ST/ER), DinR, MucA, UmuD, SamA, ImpA, CI, and the phage 22 repressor (P22 R) using a program based on algorithms of von Heijne(34) . The program allows the search for putative SPase cleavage sites with windows of variable length. The amino acid sequences shown correspond with the largest sequence giving a positive score, preferably higher than 3.5. Scores for self-cleavage sites that have been determined experimentally (46, 47, 48) are shown in boldface. Putative SPase cleavage sites coinciding with (putative) sites for self-cleavage in LexA-like proteases are marked ().




DISCUSSION

In the present paper we report the mapping of six functionally important residues of the type I SPase SipS of B. subtilis. These are serine 43, methionine 44, lysine 83, arginine 84, aspartic acid 146, and aspartic acid 153. Our data indicate that arginine 84 and aspartic acid 146 are important conformational determinants of SipS, because their replacement by other residues rendered the mutant proteins sensitive to proteolysis. What, however, could be the precise function of serine 43, lysine 83, and aspartic acid 153? Possible answers may be derived from our novel observation that SipS shares similarities with LexA-like proteases.

Self-cleavage of LexA is an intramolecular process, catalyzed by serine 119 and lysine 156(37) . The hydroxyl group of the side chain of serine 119 is the nucleophile that attacks the carbonyl carbon of the scissile peptide bond. Thus, the self-cleavage reaction of LexA would proceed through a covalent tetrahedral intermediate and an acyl-enzyme intermediate, as shown for the hydrolysis of peptide bonds by serine proteases(40) . However, the proposed mechanism for LexA differs from that of the classical serine proteases because general-base catalysis is carried out by a lysine side chain in LexA (lysine 156) instead of the imidazole group of histidine in classical serine proteases. Based on the present data, we propose that SipS also makes use of a serine-lysine catalytic dyad: the hydroxyl group of the side chain of serine 43 of SipS would act as the nucleophile that attacks the carbonyl carbon of the scissile peptide bond at the SPase cleavage site of secretory precursors. The unprotonated form of the lysine 83 -amino group would serve to activate the hydroxyl group of serine 43 and to transfer a proton from the latter group to the leaving amino group of the mature protein. This would result in the scission of the amide bond at the SPase cleavage site. As proposed for LexA, residues in close proximity of serine 43 and lysine 83 (e.g. glycine 41, methionine 44, and arginine 84) might form structural elements of the active site that are important for substrate (c-region) binding. This model is supported by several observations. First, mutants of SipS in which serine 43 was replaced by other residues only retain some residual activity when the side chain of the residue replacing serine 43 contains a hydroxyl group (threonine) or a sulfhydryl group (cysteine). The latter group is expected to react chemically in way similar to the hydroxyl group, and it is known to act as a nucleophile in thiol proteases(40) . SipS is inactive when the side chain of the residue at position 43 only contains a methyl group (alanine and valine). Interestingly, serine 119 of LexA could also be replaced by cysteine without complete loss of self-cleavage activity(41) . Second, the finding that the SipS mutants K83A, K83H, and K83R were enzymatically inactive is in accordance with the observation that the LexA mutants K156A and K156H were defective in self-cleavage and that self-cleavage of the LexA mutant K156R was highly reduced(38, 41) . Finally, the activity of SipS was not affected by inhibitors of serine proteases(42) . Similarly, self-cleavage of LexA was hardly inhibited by serine protease inhibitors, and specific binding of one inhibitor to serine 119 could only be demonstrated at very high concentrations(41, 43) . The insensitivity for inhibitors of serine proteases is a feature that SipS shares with Lep (EC) and eukaryotic type I SPases(14) .

Since serine 43 and lysine 83 of SipS are conserved in all known type I SPases of prokaryotes and the mitochondrial inner membrane, we propose that all these enzymes make use of a serine-lysine catalytic dyad. This is strongly supported by studies on the mode of action of Lep (EC) and Imp2p. First, a Lep (EC) mutant in which serine 90, the equivalent of serine 43 of SipS, was replaced by alanine was inactive(36) . Serine 90 of Lep (EC) could, however, be replaced by cysteine with only a minor reduction of activity(44) . Second, the activity of the purified S90C mutant of Lep (EC) could be inhibited by N-ethylmaleimide (44) . Third, a mutant of Imp2p in which serine 41, the equivalent of serine 43 of SipS, was replaced by alanine was inactive(18) . Fourth, the replacement of lysine 145 of Lep (EC), which is the equivalent of lysine 83 of SipS, by other residues, such as alanine, histidine, and methionine, resulted in complete inactivity of the enzyme(44, 45) . Fifth, the possibility that Lep (EC) makes use of catalytic mechanisms as defined for cysteine, aspartic, or metalloproteases could be ruled out by the fact that none of the cysteine, aspartic acid, or histidine residues of Lep (EC) were essential for activity(13, 36, 44, 45) . Although aspartic acid 153 of Lep (EC) appeared to be critical for activity in an initial study(36) , it was later shown that this residue could be replaced by asparagine without loss of activity, indicating that this residue is not directly involved in catalysis(44, 45) . This is in line with our finding that aspartic acid 91 of SipS, which corresponds to aspartic acid 153 of Lep (EC), is not important for activity of SipS.

Interestingly, neither in SipS nor in Lep (EC) (44) and LexA (38) could the putative active site lysine residue be replaced by histidine without complete loss of activity. Although this is not surprising considering the very different structure of lysine and histidine, this raises the question how the structurally related eukaryotic type I SPases Sec11, Spc18, and Spc21 achieve signal peptide cleavage; the latter enzymes appear to have a conserved histidine residue instead of the putative catalytic lysine residue of the prokaryotic and mitochondrial type I SPases (Fig. 1)(15) .

At present, we can only speculate about the function of aspartic acid 153 of SipS. A critical role of this residue in catalysis seems unlikely. First, the corresponding aspartic acid residue 280 of Lep (EC) is not critical for activity(36) . Second, unlike the classical serine proteases, LexA does not seem to require a negatively charged residue, such as aspartic acid, for catalysis(37) . Therefore, it seems unlikely that aspartic acid 153 would perform such a function in SipS. It is, however, conceivable that this residue is required for a specific feature of SPases. Possibly, aspartic acid 153 is part of an important structural element, which might also include other residues of the conserved region E of SipS. The latter hypothesis is supported by several observations. First, aspartic acid 146 appeared to be an important conformational determinant of SipS. Second, replacement of glycine 145 by alanine caused a drastic reduction of SipS activity. Glycine residues often play an important role in protein architecture. Finally, asparagine 147, serine 151, and arginine 155 appeared to be important for optimal activity of SipS.

In conclusion, the present data provide strong evidence that prokaryotic type I SPases use a serine-lysine catalytic dyad for signal peptide cleavage. This model is particularly attractive because serine 43 of SipS and its equivalents in other type I SPases are located at the outside of the cytoplasmic membrane, in close proximity to the membrane surface(14, 15) , where c-regions of exported precursors are likely to emerge from the translocation apparatus.


FOOTNOTES

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

§
Present address: Dept. of Biochemistry, Biocenter of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.: 41612672172; Fax.: 41612672148; dijl{at}ubaclu.unibas.ch.

Supported by Gist-brocades B.V. (Delft, The Netherlands) and in part by a CEC Biotech Grant BIO2 CT 920254.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; SPase, signal peptidase; Lep, leader peptidase; EC, Escherichia coli; ST, Salmonella typhimurium; PF, Pseudomonas fluorescens; SipS, signal peptidase of B. subtilis; Imp, inner membrane protease; SPC, signal peptidase complex; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. M. Sarvas and U. Airaksinen for preparing the SipS antiserum, Henk Mulder for preparing figures, and A. Nauta and B. J. Haijema for useful discussions.


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