Inhibition of purified Escherichia coli leader peptidase by the leader (signal) peptide of bacteriophage M13 procoat.

The leader peptide of bacteriophage M13 procoat inhibited the cleavage of M13 procoat or pre-maltose-binding protein by purified Escherichia coli leader peptidase. This finding confirms inferences that the leader is the primary site of enzyme recognition and suggests a rationale for the rapid hydrolysis of leader peptides in vivo.     

Reconstitution of rapid and asymmetric assembly of M13 procoat protein into liposomes which have bacterial leader peptidase

The leader peptidase of Escherichia coli cleaves a 23-residue leader sequence from M13 procoat to yield mature coat protein in virus-infected cells. We have reconstituted pure leader peptidase into vesicles of E. coli lipids and found that these liposomes are active in the conversion of procoat to coat. Trypsin removes all but 10% of the leader peptidase, yet the vesicles retain nearly full capacity to convert procoat to coat, suggesting that only procoat which inserts across the liposomal membrane is a substrate for leader peptidase. This is confirmed by the finding that over 70% of the coat protein produced by these liposomes spans the membrane. The rate at which leader peptidase inside protease-treated liposomes cleaves externally added procoat is comparable to the rate of procoat cleavage by the same amount of leader peptidase in detergent micelles. Thus, procoat can rapidly integrate across a liposomal membrane and be cleaved to coat protein. These findings confirm the central part of the membrane trigger hypothesis that certain proteins (such as procoat) can cross a bilayer without the aid of a proteinaceous pore or transport system.     

Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.     

The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical.

Proteins which are transported across the bacterial plasma membrane, endoplasmic reticulum and thylakoid membrane are usually synthesized as larger precursors containing amino-terminal targeting signals. Removal of the signals is carried out by specific, membrane-bound processing peptidases. In this report we show that the reaction specificities of these three peptidases are essentially identical. Precursors of two higher plant thylakoid lumen proteins are efficiently processed by purified Escherichia coli leader peptidase. Processing of one precursor, that of the 23 kd photosystem II protein, by both the thylakoidal and E. coli enzymes generates the correct mature amino terminus. Similarly, leader (signal) peptides of both eukaryotic and prokaryotic origin are cleaved by partially purified thylakoidal processing peptidase. No evidence of incorrect processing was obtained. Both leader peptidase and thylakoidal peptidase are inhibited by a synthetic leader peptide.     

X-ray structure and site-directed mutagenesis analysis of the Escherichia coli colicin M immunity protein

Colicin M (ColM), which is produced by some Escherichia coli strains to kill competitor strains from the same or related species, was recently shown to inhibit cell wall peptidoglycan biosynthesis through enzymatic degradation of its lipid II precursor. ColM-producing strains are protected from the toxin that they produce by coexpression of a specific immunity protein, named Cmi, whose mode of action still remains to be identified. We report here the resolution of the crystal structure of Cmi, which is composed of four β strands and four α helices. This rather compact structure revealed a disulfide bond between residues Cys31 and Cys107. Interestingly, these two cysteines and several other residues appeared to be conserved in the sequences of several proteins of unknown function belonging to the YebF family which exhibit 25 to 35% overall sequence similarity with Cmi. Site-directed mutagenesis was performed to assess the role of these residues in the ColM immunity-conferring activity of Cmi, which showed that the disulfide bond and residues from the C-terminal extremity of the protein were functionally essential. The involvement of DsbA oxidase in the formation of the Cmi disulfide bond is also demonstrated.     

Alteration by site-directed mutagenesis of the conserved lysine residue in the consensus ATP-binding sequence of the RecB protein of Escherichia coli.

The RecB and RecD subunits of the RecBCD enzyme of Escherichia coli contain amino acid sequences similar to a consensus mononucleotide binding motif found in a large number of other enzymes. We have constructed by site-directed mutagenesis a lysine-to-glutamine mutation in this sequence in the RecB protein. The mutant enzyme (RecB-K29Q-CD) has essentially no nuclease or ATP hydrolysis activity on double-stranded DNA, showing the importance of RecB for unwinding double-stranded DNA. However, ATP hydrolysis stimulated by single-stranded DNA is reduced by only about 5-8-fold compared to the wild-type, nuclease activity on single-stranded DNA is reduced by less than 2-fold, and the nuclease activity of the RecB-K29Q-CD enzyme requires ATP. The effects of the RecB mutation suggest that the RecD protein hydrolyzes ATP and can stimulate the RecBCD enzyme nuclease activity on single-stranded DNA.     

Functional analysis of the Escherichia coli molybdopterin cofactor biosynthesis protein MoeA by site-directed mutagenesis

Five moeA mutants were generated by replacing some conserved amino acids of MoeA by site-directed mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Mo-molybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterin-derived dephospho FormA as compared to bacteria transformed with wild-type moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.     

Viable transmembrane region mutants of bacteriophage M13 coat protein prepared by site-directed mutagenesis

Bacteriophage M13 coat protein - a 50-residue protein located at the E. coli host membrane during phage reproduction - is subjected to cytoplasmic, membrane-bound, and DNA-interactive environments during the phage life cycle. In research to examine the specific features of primary/secondary structure in the effective transmembrane (TM) region of the protein (residues 21-39: YIGYAWAMVVVIVGATIGI) which modulate its capacity to respond conformationally to the progressive influences of these varying environments, we have prepared over two dozen viable mutant phages with alterations in their coat protein TM regions. Mutants were obtained through use of site-directed mutagenesis techniques in combination with three "randomized" oligonucleotides which spanned the TM region. No subcloning was required. Among mutations observed were those in which each of the four TM Val residues was changed to Ala, and several with increased Ser or Thr content, including one double Ser mutant (G23S-A25S). Polar substitutions arising at Gly23 and Tyr24-including G23D, Y24H, Y24D and Y24N-suggested that this local segment resides external to the host membrane. Milligram quantities of mutant coat proteins are obtained by growing M13 mutant phages in liter preparations, with isotopic (e.g., 13C) labelling at desired sites, for subsequent characterization and conformational analysis in membrane-mimetic media.     

A water-soluble form of penicillin-binding protein 2 of Escherichia coli constructed by site-directed mutagenesis

Penicillin-binding protein (PBP) 2 of Escherichia coli is located in the cytoplasmic membrane. The N-terminal hydrophobic segment (31 amino acids, residues 15-45) of PBP2 was removed by a deletion in the PBP2 gene by site-directed mutagenesis, resulting in the production of a water-soluble form of PBP2 (called PBP2*). PBP2* retained the penicillin-binding activity, was localized in the cytoplasm and was overproduced under the control of the lpp-lac promoter. this indicates that the removed hydrophobic segment is an uncleaved signal sequence required for translocation of PBP2 across the cytoplasmic membrane, and also suggests that the segment anchors the protein to the membrane.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

Mitochondrial inner membrane protease 1 of Saccharomyces cerevisiae shows sequence similarity to the Escherichia coli leader peptidase

Summary The nuclear yeast mutant pet ts2858 is defective in the removal of pre-sequences from the mitochondrially encoded cytochrome oxidase subunit II (COXII) and the processing intermediate of cytochrome b ₂ (Cytb ₂), a nuclear gene product. In order to identify the genetic lesion in this mutant we have cloned and characterized a DNA region which complements the pet ts2858 mutation. The DNA sequence revealed three open reading frames, one of which is responsible for the complementation. A 570 by reading frame represents the structural gene PET2858, as demonstrated by in vitro mutagenesis, gene expression from a foreign promoter, and allelism tests. PET2858 encodes a 21.4 kDa protein, which is essential for growth on non-fermentable carbon sources and for the proteolytic processing of COXII and the Cytb ₂ intermediate. When the N-terminus of the PET2858 protein is fused to a reporter protein, the resulting hybrid molecule is imported into mitochondria. Interestingly, the N-terminal half of the deduced PET2858 protein exhibits 30.7% amino acid identity to the leader peptidase of Escherichia coli. These results suggest that PET2858 codes for a mitochondrial inner membrane protease (IMP1) or at least a subunit of it. This protease is involved in protein processing and export from the mitochondrial matrix.Dedicated to Professor Dr. Peter Starlinger on the occasion of his 60th birthday     

Identification of potential active-site residues in the Escherichia coli leader peptidase.

Leader peptidase of Escherichia coli cleaves the leader sequence from the amino terminus of membrane and secreted proteins after these proteins insert across the membrane. Despite considerable research, the mechanism of catalysis of leader peptidase remains unknown. This peptidase cannot be classified using protease inhibitors to the serine, cysteine, aspartic acid, or metallo- classes of proteases (Zwizinski, C., Date, T., and Wickner, W. (1981) J. Biol. Chem. 256, 3593-3597). Using site-directed mutagenesis, we have attempted to place leader peptidase in one of these groups. We found that leader peptidase, lacking all of the cysteine residues, can cleave the leader peptide from procoat, the precursor to bacteriophage M13 coat protein. Replacement of each histidine residue with an alanyl residue was without effect on catalysis. Among all the serine and aspartic acid residues, serine 90 and serine 185 as well as aspartic acid 99, 153, 273, and 276 are necessary to cleave procoat in a detergent extract. However, only serine 90 and aspartic acid 153 were required for processing using a highly sensitive in vivo assay. In addition to the residues directly affecting catalysis, aspartic acid 99 plays a role in maintaining the structure of leader peptidase. Replacement of this residue with alanine results in a very unstable leader peptidase protein. This study thus defines two critical residues, serine 90 and aspartic acid 153, that may be directly involved in catalysis and provides evidence that leader peptidase belongs to a novel class of serine proteases.     

Mapping of catalytically important domains in Escherichia coli leader peptidase.

Leader peptidase (Lep) is a central component of the secretory machinery of Escherichia coli, where it serves to remove signal peptides from secretory proteins. It spans the inner membrane twice with a large C-terminal domain protruding into the periplasmic space. To investigate the importance of the different structural domains for the catalytic activity, we have studied the effects of a large panel of Lep mutants on the processing of signal peptides, both in vivo and in vitro. Our data suggest that the first transmembrane and cytoplasmic regions are not directly involved in catalysis, but that the second transmembrane region and the region immediately following it may be in contact with the signal peptide and/or located spatially close to the active site of Lep.     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.     

Conditionally lethal amber mutations in the leader peptidase gene of Escherichia coli.

The lep gene of Escherichia coli encodes the leader peptidase which cleaves amino-terminal leader sequences of secreted proteins. To facilitate the study of structure-function relationships of the leader peptidase, 22 amber mutations in lep were isolated by localized mutagenesis. These amber mutants grew at 32 degrees C but not at 42 degrees C in the presence of a temperature-sensitive amber suppressor. Most of them were lethal under sup0 conditions. However, one amber mutant, the lep-9 mutant, exhibited temperature-sensitive growth in the sup0 strain, indicating that the amber fragment is active at 32 degrees C but not at 42 degrees C. Protein precursors of the maltose-binding protein and OmpA accumulate strikingly in the lep-9 mutant.     

In vitro insertion of leader peptidase into Escherichia coli membrane vesicles.

Leader peptidase is an integral protein of the Escherichia coli cytoplasmic membrane whose topology is known. We have taken advantage of this knowledge and available mutants of this enzyme to develop a genetic test for a cell-free protein translocation reaction. We report that leader peptidase inserted into inverted plasma membrane vesicles in its correct transmembrane orientation. We have examined the in vitro membrane assembly characteristics of a variety of leader peptidase mutants and found that domains required for insertion in vivo are also necessary for insertion in vitro. These data demonstrate the physiological validity of the in vitro insertion reaction and strengthen the use of this in vitro protein translocation reaction for the dissection of this complex sorting pathway.     

Characterization of the internal signal-anchor domain of Escherichia coli leader peptidase

Leader peptidase, an integral transmembrane protein of Escherichia coli, is synthesized without a cleavable amino-terminal leader peptide. Of the five domains that participate in the membrane assembly of this protein, one is an internal "signal" region. We have used oligonucleotide-directed mutagenesis to examine the properties of the internal signal that are crucial for leader peptidase assembly. For this purpose, the net charge at the amino terminus of the internal signal was changed from +2 to +1 and -1 and, at the carboxyl terminus of the signal, from 0 to -1 or +1. These mutations had no effect on the membrane assembly of leader peptidase, suggesting that the charges have little role in the signal function. The apolar core of this signal was disrupted by substitution of basic amino acids for apolar residues. Substitution of an arginyl residue at position 70, or two arginyl residues at position 67 and 69, prevented membrane assembly. However, substitution of an arginyl residue at position 66 or either arginyl or lysyl residue at position 68 was without effect. Thus, while the apolar character of the internal signal is important, the precise position of a charged residue determines its effect on assembly.