(Received for publication, September 16, 1994; and in revised form, December 5, 1994)
From the
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.
The most important common feature of protein transport across
the cytoplasmic membrane of prokaryotes and the endoplasmic reticular
(ER) ()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 () 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) .
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--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)--lactamase processing by
SipS mutants. Processing of pre(A13i)-
-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)-
-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)--lactamase processing by SipS mutants with severely
reduced activity. Processing of pre(A13i)-
-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)-
-lactamase in the presence of
wild-type SipS (E. coli C600 (pGDL41)); -, processing of
pre(A13i)-
-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)--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.
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)--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)--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)-
-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.
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
(
).
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.