Cloning, expression, and purification of functional Sec11a and Sec11b, type I signal peptidases of the archaeon Haloferax volcanii

Across evolution, type I signal peptidases are responsible for the cleavage of secretory signal peptides from proteins following their translocation across membranes. In Archaea, type I signal peptidases combine domain-specific features with traits found in either their eukaryal or bacterial counterparts. Eukaryal and bacterial type I signal peptidases differ in terms of catalytic mechanism, pharmacological profile, and oligomeric status. In this study, genes encoding Sec11a and Sec11b, two type I signal peptidases of the halophilic archaeon Haloferax volcanii, were cloned. Although both genes are expressed in cells grown in rich medium, gene deletion approaches suggest that Sec11b, but not Sec11a, is essential. For purification purposes, tagged versions of the protein products of both genes were expressed in transformed Haloferax volcanii, with Sec11a and Sec11b being fused to a cellulose-binding domain capable of interaction with cellulose in hypersaline surroundings. By employing an in vitro signal peptidase assay designed for use with high salt concentrations such as those encountered by halophilic archaea such as Haloferax volcanii, the signal peptide-cleaving activities of both isolated membranes and purified Sec11a and Sec11b were addressed. The results show that the two enzymes differentially cleave the assay substrate, raising the possibility that the Sec11a and Sec11b serve distinct physiological functions.     

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.     

Type I signal peptidase: an overview

The signal hypothesis suggests that proteins contain information within their amino acid sequences for protein targeting to the membrane. These distinct targeting sequences are cleaved by specific enzymes known as signal peptidases. There are various type of signal peptidases known such as type I, type II, and type IV. Type I signal peptidases are indispensable enzymes, which catalyze the cleavage of the amino-terminal signal-peptide sequences from preproteins, which are translocated across biological membranes. These enzymes belong to a novel group of serine proteases, which generally utilize a Ser-Lys or Ser-His catalytic dyad instead of the prototypical Ser-His-Asp triad. Despite having no distinct consensus sequence other than a commonly found 'Ala-X-Ala' motif preceding the cleavage site, signal sequences are recognized by type I signal peptidase with high fidelity. Type I signal peptidases have been found in bacteria, archaea, fungi, plants, and animals. In this review, I present an overview of bacterial type I signal peptidases and describe some of their properties in detail.     

The role of the conserved box E residues in the active site of the Escherichia coli type I signal peptidase

Type I signal peptidases are integral membrane proteins that function to remove signal peptides from secreted and membrane proteins. These enzymes carry out catalysis using a serine/lysine dyad instead of the prototypical serine/histidine/aspartic acid triad found in most serine proteases. Site-directed scanning mutagenesis was used to obtain a qualitative assessment of which residues in the fifth conserved region, Box E, of the Escherichia coli signal peptidase I are critical for maintaining a functional enzyme. First, we find that there is no requirement for activity for a salt bridge between the invariant Asp-273 and the Arg-146 residues. In addition, we show that the conserved Ser-278 is required for optimal activity as well as conserved salt bridge partners Asp-280 and Arg-282. Finally, Gly-272 is essential for signal peptidase I activity, consistent with it being located within van der Waals proximity to Ser-278 and general base Lys-145 side-chain atoms. We propose that replacement of the hydrogen side chain of Gly-272 with a methyl group results in steric crowding, perturbation of the active site conformation, and specifically, disruption of the Ser-90/Lys-145 hydrogen bond. A refined model is proposed for the catalytic dyad mechanism of signal peptidase I in which the general base Lys-145 is positioned by Ser-278, which in turn is held in place by Asp-280.     

The chemistry and enzymology of the type I signal peptidases

The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membrane-bound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes. These peptidases are termed type I signal (or leader) peptidases. They share the unusual feature of being resistant to the general inhibitors of the four well-characterized peptidase classes. The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family. This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases.     

Biochemical characterization of signal peptidase I from gram-positive Streptococcus pneumoniae

Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive Streptococcus pneumoniae. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of S. pneumoniae signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of S. pneumoniae signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases and Escherichia coli signal peptidase I. Similar to LexA-like proteases, S. pneumoniae signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.     

Type I signal peptidases of Gram-positive bacteria

Proteins that are exported from the cytoplasm to the periplasm and outer membrane of Gram-negative bacteria, or the cell wall and growth medium of Gram-positive bacteria, are generally synthesized as precursors with a cleavable signal peptide. During or shortly after pre-protein translocation across the cytoplasmic membrane, the signal peptide is removed by signal peptidases. Importantly, pre-protein processing by signal peptidases is essential for bacterial growth and viability. This review is focused on the signal peptidases of Gram-positive bacteria, Bacillus and Streptomyces species in particular. Evolutionary concepts, current knowledge of the catalytic mechanism, substrate specificity requirements and structural aspects are addressed. As major insights in signal peptidase function and structure have been obtained from studies on the signal peptidase LepB of Escherichia coli, similarities and differences between this enzyme and known Gram-positive signal peptidases are highlighted. Notably, while the incentive for previous research on Gram-positive signal peptidases was largely based on their role in the biotechnologically important process of protein secretion, present-day interest in these essential enzymes is primarily derived from the idea that they may serve as targets for novel anti-microbials.     

Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. DeltaN54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191) Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.     

Two subunits of the canine signal peptidase complex are homologous to yeast SEC11 protein

Canine microsomal signal peptidase activity was previously isolated as a complex of five subunits (25, 22/23, 21, 18, and 12 kDa). Two of the signal peptidase complex (SPC) subunits (23/23 and 21 kDa) have been cloned and sequenced. One of these, the 21-kDa subunit, was observed to be a mammalian homolog of SEC11 protein (Sec11p) (Greenburg, G., Shelness, G. S., and Blobel, G. (1989) J. Biol. Chem. 264, 15762-15765) a gene product essential for signal peptide processing and cell growth in yeast (B?hni, P.C., Deshqies, R.J., and Schekman, R.W. (1988) J. Cell Biol. 106, 1035-1042). cDNA clones for the 18-kDa SPC subunit have now been characterized and found to encode a second SEC11p homolog. Both the 18- and 21-kDa canine SPC subunits are integral membrane proteins by virtue of their resistance to alkaline extraction. Upon detergent solubilization, both proteins are found in a complex with the 22/23 kDa SPC subunit, the only SPC subunit containing N-linked oligosaccharide. No steady-state pool of canine Sec11p-like monomers is detected in microsomal membranes. Alkaline extraction of microsomes prior to solubilization or solubilization at alkaline pH causes partial dissociation of the SPC. The Sec11p-like subunits displaced from the complex under these conditions demonstrate no signal peptide processing activity by themselves. The existence of homologous subunits is common to a number of known protein complexes and provides further evidence that the association between SPC proteins observed in vitro may be physiologically relevant to the mechanism of signal peptide processing and perhaps protein translocation.     

Crystal structure of Bacillus subtilis signal peptide peptidase A

Signal peptide peptidase A (SppA) is a membrane-bound self-compartmentalized serine protease that functions to cleave the remnant signal peptides left behind after protein secretion and cleavage by signal peptidases. SppA is found in plants, archaea and bacteria. Here, we report the first crystal structure of a Gram-positive bacterial SppA. The 2.4-Å-resolution structure of Bacillus subtilis SppA (SppA_BS) catalytic domain reveals eight SppA_BS molecules in the asymmetric unit, forming a dome-shaped octameric complex. The octameric state of SppA_BS is supported by analytical size-exclusion chromatography and multi-angle light scattering analysis. Our sequence analysis, mutagenesis and activity assays are consistent with Ser147 serving as the nucleophile and Lys199 serving as the general base; however, they are located in different region of the protein, more than 29 Å apart. Only upon assembling the octamer do the serine and lysine come within close proximity, with neighboring protomers each providing one-half of the catalytic dyad, thus producing eight separate active sites within the complex, twice the number seen within Escherichia coli SppA (SppA_EC). The SppA_BS S1 substrate specificity pocket is deep, narrow and hydrophobic, but with a polar bottom. The S3 pocket, which is constructed from two neighboring proteins, is shallower, wider and more polar than the S1 pocket. A comparison of these pockets to those seen in SppA_EC reveals a significant difference in the size and shape of the S1 pocket, which we show is reflected in the repertoire of peptides the enzymes are capable of cleaving.     

SEC11 is required for signal peptide processing and yeast cell growth

Among the collection of temperature-sensitive secretion mutants of Saccharomyces cerevisiae, sec11 mutant cells are uniquely defective in signal peptide processing of at least two different secretory proteins. At 37 degrees C, the restrictive growth temperature, sec11 cells accumulate core-glycosylated forms of invertase and acid phosphatase, each retaining an intact signal peptide. In contrast, other sec mutant strains in which transport of core-glycosylated molecules from the endoplasmic reticulum is blocked show no defect in signal peptide cleavage. A DNA fragment that complements the sec11-7 mutation has been cloned. Genetic analysis indicates that the complementing clone contains the authentic SEC11 gene, and that a null mutation at the SEC11 locus is lethal. The DNA sequence of SEC11 predicts a basic protein (estimated pI of 9.5) of 167 amino acids including an NH2-terminal hydrophobic region that may function as a signal and/or membrane anchor domain. One potential N-glycosylation site is found in the 18.8-kD (Sec 11p) predicted protein. The mass of the SEC11 protein is very close to that found for two of the subunits of the canine and hen oviduct signal peptidases. Furthermore, the chromatographic behavior of the hen oviduct enzyme indicates an overall basic charge comparable to the predicted pI of the Sec11p.     

Structure and mechanism of Escherichia coli type I signal peptidase

Type I signal peptidase is the enzyme responsible for cleaving off the amino-terminal signal peptide from proteins that are secreted across the bacterial cytoplasmic membrane. It is an essential membrane bound enzyme whose serine/lysine catalytic dyad resides on the exo-cytoplasmic surface of the bacterial membrane. This review discusses the progress that has been made in the structural and mechanistic characterization of Escherichia coli type I signal peptidase (SPase I) as well as efforts to develop a novel class of antibiotics based on SPase I inhibition. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Identification of residues essential for the catalytic activity of Sec11b, one of the two type I signal peptidases of Haloferax volcanii

Sec11b is one of two signal peptidases (SPases) in the haloarchaeon Haloferax volcanii. Site-directed mutagenesis revealed Ser-72, His-137 and Asp-187 as essential for signal peptide cleavage. Thus, like the SPase of the methanoarchaeon Methanococcus voltae, H. volcanii Sec11b uses a catalytic mechanism reminiscent of its eukaryal rather than its bacterial counterpart. The availability of an additional model system to study the archaeal SPase, now in the form of the purified protein, promises additional insight into the behavior of this enzyme.     

Genetic complementation in yeast reveals functional similarities between the catalytic subunits of mammalian signal peptidase complex

Type I signal peptidases (SPs) comprise a family of structurally related enzymes that cleave signal peptides from precursor proteins following their transport out of the cytoplasmic space in eukaryotic and prokaryotic cells. One such enzyme, the mitochondrial inner membrane peptidase, has two catalytic subunits, which recognize distinct cleavage site motifs in their signal peptide substrates. The only other known type I SP with two catalytic subunits is the signal peptidase complex (SPC) in the mammalian endoplasmic reticulum. Here, we tested the hypothesis that, as with inner membrane peptidase catalytic subunits, SPC catalytic subunits exhibit nonoverlapping substrate specificity. We constructed two yeast strains without endogenous SP, one expressing canine SPC18 and the other expressing a truncation of canine SPC21 (SPC21 Delta N), which lacks 24 N-terminal residues that prevent expression of SPC21 in yeast. By monitoring a variety of soluble and membrane-bound substrates, we find that, in contrast to the tested hypothesis, SPC catalytic subunits exhibit overlapping substrate specificity. SPC18 and SPC21 Delta N do, however, cleave some substrates with different efficiencies, although no pattern for this behavior could be discerned. In light of the functional similarities between SPC proteins, we developed a membrane protein fragmentation assay to monitor the position of the catalytic sites relative to the surface of the endoplasmic reticulum membrane. Using this assay, our results suggest that the active sites of SPC18 and SPC21 Delta N are located 4-11 A above the membrane surface. These data, thus, support a model that SPC18 and SPC21 are functionally and structurally similar to each other.     

Compensatory effect of the minor Streptomyces lividans type I signal peptidases on the SipY major signal peptidase deficiency as determined by extracellular proteome analysis

The developmentally complex bacterium Streptomyces lividans has the ability to produce and secrete a significant amount of protein and possesses four different type I signal peptidase genes (sipW, sipX, sipY and sipZ) that are unusually clustered in its chromosome. 2-DE and subsequent MS of extracellular proteins showed that proteins with typical export signals for type I and type II signal peptidases are the main components of the S. lividans secretome. Secretion of extracellular proteins is severely reduced in a strain deficient in the major type I signal peptidase (SipY). This deficiency was efficiently compensated by complementation with any of the other three signal peptidases as deduced from a comparison of the corresponding 2-D PAGE patterns with that of the wild-type strain.     

Factors governing nonoverlapping substrate specificity by mitochondrial inner membrane peptidase

At least three peptidases are involved in cleaving presequences from imported mitochondrial proteins. One of the peptidase, the inner membrane peptidase, has two catalytic subunits, Imp1p and Imp2p, which are structurally related but functionally distinct in the yeast Saccharomyces cerevisiae. Whereas both subunits are members of the type I signal peptidase family, they exhibit nonoverlapping substrate specificities. A clue to the substrate specificity mechanism has come from our discovery of the importance not only of the -1 and -3 residues in the signal peptides cleaved by Imp1p and Imp2p but also the +1 cargo residues attached to the signal peptides. We specifically find that Imp1p prefers substrates having a negatively charged residue (Asp or Glu) at the +1 position, whereas Imp2p prefers substrates having the Met residue at the +1 position. We further suggest that the conformation of the cargo is important for substrate recognition by Imp2p. A role for the cargo in presequence recognition distinguishes Imp1p and Imp2p from other type I signal peptidases.     

An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei

The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival.     

The peptidases from fungi and viruses

Fungi and viruses encode a variety of peptidases having a plethora of functions. Many fungal peptidases are extracellular and are likely used to degrade proteins in their environment. Viral peptidases are processing enzymes, intimately involved in the virus infectious cycle. The viral RNA genome is translated by the host-cell machinery into a large polyprotein that is cleaved by the viral peptidases into mature capsid proteins, non-structural proteins and enzymes. I review the structure and catalytic mechanism of scytalidoglutamic peptidase isolated from the wood-destroying fungus Scytalidium lignicolum. This enzyme has a unique beta-sandwich fold and a novel catalytic mechanism based on a glutamate, a glutamine and a nucleophilic water molecule. Hepatitis A virus (HAV) 3C peptidase was the first structure identified for a viral 3C enzyme that exhibited the three-dimensional fold of the chymotrypsin family of serine peptidases but had a cysteine sulfur atom instead of the serine oxygen as the nucleophile. The structure of HAV 3C was unusual in that the Asp residue expected as the third member of the catalytic triad did not interact with the general base His. The present structure is of a beta-lactone-inhibited version of HAV 3C that has a restored catalytic triad.     

Signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p of the mitochondrial inner membrane protease

We have performed a site-directed mutagenesis study showing that residues comprising the type I signal peptidase signature in the two catalytic subunits of the yeast inner membrane protease, Imp1p and Imp2p, are functionally important, consistent with the idea that these subunits contain a serine/lysine catalytic dyad. Previous studies have shown that Imp1p cleaves signal peptides having asparagine at the -1 position, which deviates from the typical signal peptide possessing a small uncharged amino acid at this position. To determine whether asparagine is responsible for the nonoverlapping substrate specificities exhibited by the inner membrane protease subunits, we have substituted asparagine with 19 amino acids in the Imp1p substrate i-cytochrome (cyt) b(2). The resulting signal peptides containing alanine, serine, cysteine, leucine, and methionine can be cleaved efficiently by Imp1p. The remaining mutant signal peptides are cleaved inefficiently or not at all. Surprisingly, none of the amino acid changes results in the recognition of i-cyt b(2) by Imp2p, whose natural substrate, i-cyt c(1), has alanine at the -1 position. The data demonstrate that (i) although the -1 residue is important in substrates recognized by Imp1p, signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p, and (ii) a -1 asparagine does not govern the substrate specificity of the inner membrane protease subunits.     

Regulation of signal peptidase by phospholipids in membrane: characterization of phospholipid bilayer incorporated Escherichia coli signal peptidase

Prokaryotic signal peptidases are membrane-bound enzymes. They cleave signal peptides from precursors of secretary proteins. To study the enzyme in its natural environment, which is phospholipid bilayers, we developed a method that allows us effectively to incorporate full-length Escherichia coli signal peptidase I into phospholipid vesicles. The membrane-bound signal peptidase showed high activity on a designed substrate. The autolysis site of the enzyme is separated from its catalytic site in vesicles by the lipid bilayer, resulting in a dramatic decrease of the autolysis rate. Phosphotidylethanolamine, which is the most abundant lipid in Escherichia coli inner membrane, is required to maintain activity of the membrane-incorporated signal peptidase. The maximal activity is achieved at about 55% phosphotidylethanolamine. Negatively charged lipids, which are also abundant in Escherichia coli inner membrane, enhances the activity of the enzyme too. Its mechanism, however, cannot be fully explained by its ability to increase the affinity of the substrate to the membrane. A reaction mechanism was developed based on the observation that cleavage only takes place when the enzyme and the substrate are bound to the same vesicle. Accordingly, a kinetic analysis is presented to explain some of the unique features of phospholipid vesicles incorporated signal peptidase, including the effect of lipid concentration and substrate-vesicle interaction.