The pro-sequence domain of streptopain directs the folding of the mature enzyme

The cysteine endopeptidase streptopain, an extracellular enzyme from pathogenic Streptococcus pyogenes, is synthesized as a precursor containing an NH2-terminal pro-sequence. The pro-sequence of streptopain was expressed in Escherichia coli and subjected to structural and functional investigation. Heat-induced denaturation of the pro-sequence studied using circular dichroism spectroscopy revealed that it forms a compact structure and represents an independently folded domain. The isolated pro-sequence exhibits high affinity towards mature streptopain and associates with its cognate enzyme by forming an equimolar complex. Refolding of denatured streptopain in the presence of pro-sequence in vitro facilitated recovery of active enzyme. Expression of the mature streptopain in E. coli either alone, or in trans with its pro-sequence as an independent polypeptide, led to the formation of insoluble protein aggregates or functionally active enzyme, respectively. These results demonstrate that the pro-sequence domain acts as an intramolecular chaperone that directs the correct folding of the mature streptopain.     

Germination-specific cortex-lytic enzymes from Clostridium perfringens S40 spores: time of synthesis, precursor structure and regulation of enzymatic activity

Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation. The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence. The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination. A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.     

Production of hydrolytic enzymes by oral isolates of Eikenella corrodens

Abstract Thermus thermophilus cells harboring an expression plasmid for the aqualysin I gene secrete the mature enzyme into the medium. In an Escherichia coli expression system, a precursor of the enzyme with the C-terminal pro-sequence is accumulated in the cells, and upon treatment at 65°C the active enzyme is produced. One- to 10-amino acid residue deletions, as well as complete 105-residue deletion of the C-terminal pro-sequence from the C-terminus, did not affect the production of the enzyme in T. coli cells. T. thermophilus cells harboring plasmids for mutant precursors with one- and three-residue deletions secreted the enzyme extracellularly. However, transformants harboring plasmids for mutant precursors with deletions of five or more amino acid residues could not be obtained. These results suggest that the C-terminal pro-sequence plays an important role in the extracellular secretion of the enzyme in T. thermophilus cells.     

A non-covalent NH2-terminal pro-region aids the production of active aqualysin I (a thermophilic protease) without the COOH-terminal pro-sequence in Escherichia coli

The precursor of aqualysin I, an extracellular protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, the protease domain and a C-terminal pro-sequence. In an Escherichia coli expression system, mature and active aqualysin I is formed by treatment at 65 degrees C and the N-pro-sequence is required for its production. Complete deletion of the C-pro-sequence did not affect the production of active aqualysin I, indicating that the C-pro-sequence is not essential. A non-covalent N-pro-region was separately synthesized from the protease domain with or without the C-pro-sequence. In this system, mature and active aqualysin I was detected only when the C-pro-sequence was deleted.     

A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin-type protease) with a C-terminal extended sequence: its purification and in vitro processing

The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65°C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS-polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.     

Contribution of C-tail residues of potato carboxypeptidase inhibitor to the binding to carboxypeptidase A A mutagenesis analysis.

The role of each residue of the potato carboxypeptidase inhibitor (PCI) C-terminal tail, in the interaction with carboxypeptidase A (CPA), has been studied by the analysis of two main kinds of site-directed mutants: the point substitution of each C-terminal residue by glycine and the sequential deletions of the C-terminal residues. The mutant PCI-CPA interactions have been characterized by the measurement of their inhibition constant, Ki, in several cases, by their kinetic association and dissociation constants determined by presteady-state analysis, and by computational approaches. The role of Pro36 appears to be mainly the restriction of the mobility of the PCI C-tail. In addition, and unexpectedly, both Gly35 and Pro36 have been found to be important for folding of the protein core. Val38 has the greatest enthalpic contribution to the PCI-CPA interaction. Although Tyr37 has a minor contribution to the binding energy of the whole inhibitor, it has been found to be essential for the interaction with the enzyme following the cleavage of the C-terminal Gly39 by CPA. The energetic contribution of the PCI secondary binding site has been evaluated to be about half of the total free energy of dissociation of the PCI-CPA complex.     

Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli

Subtilisin E, an alkaline serine protease of Bacillus subtilis 168, is first produced as a precursor, pre-pro-subtilisin, which consists of a signal peptide for protein secretion (pre-sequence) and a peptide extension of 77 amino acid residues (pro-sequence) between the signal peptide and mature subtilisin. When the entire coding region for pre-pro-subtilisin E was cloned into an Escherichia coli expression vector, active mature subtilisin E was secreted into the periplasmic space. When the pre-sequence was replaced with the E. coli OmpA signal peptide, active subtilisin E was also produced. When the OmpA signal peptide was directly fused to the mature subtilisin sequence, no protease activity was detected, although this product had the identical primary structure as subtilisin E as a result of cleavage of the OmpA signal peptide and was produced at a level of approximately 10% of total cellular protein. When the OmpA signal peptide was fused to the 15th or 44th amino acid residue from the amino terminus of the pro-sequence, active subtilisin was also not produced. These results indicate that the pro-sequence of pre-pro-subtilisin plays an important role in the formation of enzymatically active subtilisin. It is proposed that the pro-sequence is essential for guiding appropriate folding of the enzymatically active conformation of subtilisin E.     

Genetic analyses of processing involving C-terminal cleavage in penicillin-binding protein 3 of Escherichia coli.

The processing of Escherichia coli penicillin-binding protein 3 (PBP 3) was investigated by gene manipulation for producing hybrid and truncated PBP 3 molecules. The hybrid PBP 3 was processed when the N-terminal 40 residues of PBP 3 were replaced by the murein lipoprotein signal peptide which lacked the cysteine residue for processing and followed by seven extra linker residues. In contrast, the PBP 3 molecules truncated at Thr-560 (28-residue deletion) or at Thr-497 (91-residue deletion) were not processed, and those truncated at Phe-576 (12-residue deletion) were processed at a greatly reduced rate. The results indicate that the C-terminal part, rather than the N-terminal part, is involved in the processing. This was supported by the result that the purified mature PBP 3 retained the complete N-terminal sequence with Met for translation initiation. The cleavage at the C-terminal region was shown by the loss of [35S]cysteine label when the cysteine-free hybrid PBP 3 joined to a cysteine-rich extra peptide tail was processed into the mature form. Confirmative assays for processing of PBP 3 were aided by a newly found prc mutant, defective in the processing involving the C-terminal region. A plasmid that directs PBP 3 truncated at Thr-560 complemented a thermosensitive PBP 3 mutation, but the truncated product was unstable in vivo. This suggests the importance of C-terminal hydrophobic regions that terminate at Leu-558 to PBP 3 functioning and the requirement of further-distal peptides for the stability of PBP 3.     

Pro-sequence and Ca2+-binding: implications for folding and maturation of Ntn-hydrolase penicillin amidase from E. coli

Penicillin amidase (PA) is a bacterial periplasmic enzyme synthesized as a pre-pro-PA precursor. The pre-sequence mediates membrane translocation. The intramolecular pro-sequence is expressed along with the A and B chains but is rapidly removed in an autocatalytic manner. In extensive studies we show here that the pro-peptide is required for the correct folding of PA. Pro-PA and PA unfold via a biphasic transition that is more pronounced in the case of PA. According to size-exclusion chromatography and limited proteolysis experiments, the inflection observed in the equilibrium unfolding curves corresponds to an intermediate in which the N-terminal domain (A-chain) still possesses native-like topology, whereas the B-chain is unfolded to a large extent. In a series of in vitro experiments with a slow processing mutant pro-PA, we show that the pro-sequence in cis functions as a folding catalyst and accelerates the folding rate by seven orders of magnitude. In the absence of the pro-domain the PA refolds to a stable inactive molten globule intermediate that has native-like secondary but little tertiary structure. The pro-sequence of the homologous Alcaligenes faecalis PA can facilitate the folding of the hydrolase domain of Escherichia coli PA when added in trans (as a separate polypeptide chain). The isolated pro-sequence has a random structure in solution. However, difference circular dichroism spectra of native PA and native PA with pro-peptide added in trans suggest that the pro-sequence adopts an alpha-helical conformation in the context of the mature PA molecule. Furthermore, our results establish that Ca2+, found in the crystal structure, is not directly involved in the folding process. The cation shifts the equilibrium towards the native state and facilitates the autocatalytic processing of the pro-peptide.     

The role of the N-terminal propeptide of the pro-aminopeptidase processing protease: refolding,processing, and enzyme inhibition

Pro-aminopeptidase processing protease (PA protease) is an extracellular zinc metalloprotease produced by Aeromonas caviae T-64 and it is classified as M04.016 according to the MEROPS database. The precursor of PA protease consists of four regions; a signal peptide, an N-terminal propeptide, a C-terminal propeptide, and the mature PA protease. The in vitro refolding of the intermediate pro-PA protease containing the C-terminal propeptide (MC) was investigated in the presence and absence of the N-terminal propeptide. The results indicate that the noncovalently linked N-terminal propeptide is able to assist in the refolding of MC. In the absence of the N-terminal propeptide, MC is trapped into a folding competent state that is converted into the active form by the addition of the N-terminal propeptide. Moreover, the N-terminal propeptide was found to form a complex with the folded MC and inhibit further processing of MC into the mature PA protease. Inhibitory activity of the purified N-terminal propeptide toward mature PA protease was also observed, and the mode of this inhibition was determined to be a mixed, noncompetitive inhibition with an associated allosteric effect.     

Mutations in the N- and C-terminal tails of potato carboxypeptidase inhibitor influence its oxidative refolding process at the reshuffling stage

A comparative study of the oxidative refolding for nine selected potato carboxypeptidase inhibitor (PCI) mutants was carried out using the disulfide quenching approach. The mutations were performed at the N- and C-terminal tails of PCI outside its disulfide stabilized central core. The differences between the refolding of wild type and mutant proteins were observed in the second phase of the refolding process, the reshuffling of disulfide bridges, although the first phase, nonspecific packing, was not greatly affected by the mutations. Point mutations at the C-tail or deletion of up to three C-terminal residues of PCI resulted in a lower efficiency of the reshuffling process. In the case of the mutants lacking five N-terminal or four or five C-terminal residues, no "native-like" form was observed after the refolding process. On the other hand, the double mutant G35P/P36G did not attain a native-like form either, although one slightly more stable species was observed after being submitted to refolding. The disulfide pairing of this species is different from that of the wtPCI native form. The differences between the refolding process of wild type and mutant forms are interpreted in the light of the new view of protein folding. The results of the present study support the hypothesis that the refolding of this small disulfide-rich protein, and others, is driven by noncovalent interactions at the reshuffling stage. It is also shown that the interactions established between the N- and C-tail residues and the core of PCI are important for the proper refolding of the protein.     

Primary structure of the human elafin precursor preproelafin deduced from the nucleotide sequence of its gene and the presence of unique repetitive sequences in the prosegment.

The human elafin gene was cloned and its entire nucleotide sequence was determined to deduce the amino acid sequence for the precursor of elafin, an elastase-specific inhibitor. The gene spans approximately 1.7 kb and is divided into 3 exons. The gene product preproelafin consists of 117 amino acids: the initiator Met, a putative 25-amino acid signal peptide, a pro-sequence of about 34 amino acids, and the C-terminal 57 amino acids for mature elafin. Possible covalent clotting of the prosegment and its physiological significance have been pointed out based on a remarkable sequence similarity between the pro-sequence and the guinea pig seminal clotting protein SVP-1.     

A new approach for alteration of protease functions: pro-sequence engineering

Several proteases, including the bacterial serine protease subtilisins, require the assistance of the N-terminal pro-sequence of precursors to produce active, mature enzymes. Upon completion of folding, the pro-sequence is autocatalytically degraded because it is not necessary for the activity or stability of folded, mature cognates of the original enzymes. Therefore, the pro-sequence functions as an intramolecular chaperone that guides correct folding of the protease domain. Interestingly, Shinde et al. proposed a new theory of "protein memory" in which an identical polypeptide can fold into an altered conformation with different secondary structure, stability and specificities through a mutated pro-sequence [Shinde et al. (1997) Nature 389:520–522]. We also showed that the autoprocessing efficiency was improved by modifications in the pro-sequence of mutant subtilisins with altered substrate specificity. Further, the pro-sequence from a subtilisin homologue was found to chaperone the intramolecular folding of denatured subtilisin. These results indicate that engineering of the pro-sequence, i.e., site-directed and/or random mutagenesis, chimeras and gene shuffling between members of the family, would be a useful method for improving the functions of autoprocessing proteases. Conventional protein engineering techniques have thus far employed mutagenesis in the protease domain to modify the enzymatic properties. This new approach, which we term "pro-sequence engineering", is not only an important tool for studying the mechanism of protein folding, but also a promising technology for creating unique proteases with various beneficial properties.     

Secondary binding site of the potato carboxypeptidase inhibitor. Contribution to its structure, folding, and biological properties

The contribution of each residue of the potato carboxypeptidase inhibitor (PCI) secondary binding site to the overall properties of this protein has been examined using alanine-scanning mutagenesis. Structural and enzymatic studies, performed on a series of PCI mutants, demonstrate that the proper positioning of the primary site for efficient binding and inhibition of carboxypeptidase A is significantly dependent on such a secondary contact region. The aromatic residues in this region play a key role in the stabilization of the PCI-enzyme complex, whereas polar residues contribute little to this task. A comparative study of the oxidative folding of these PCI mutants has been carried out using the disulfide quenching approach. The data, together with the structural characterization of some of these mutants, clearly indicate that noncovalent forces drive the refolding of this small disulfide-rich protein at the reshuffling stage, the rate-limiting step of the process. Moreover, it reveals that by introducing new noncovalent intramolecular contacts in PCI, we may create more stable variants, which also show improved folding efficiency. Taken together, the collected results clarify the folding determinants of the primary and secondary binding sites of PCI and their contribution to the inhibition of the carboxypeptidase, providing clues about PCI evolution and knowledge for its biotechnological redesign.     

Role of the COOH-terminal pro-sequence of aqualysin I (a heat-stable serine protease) in its extracellular secretion by Thermus thermophilus

Aqualysin I is a subtilisin-type serine protease secreted into the medium by Thermus aquaticus YT-1. Thermus thermophilus cells harboring a plasmid for the aqualysin I precursor secreted pro-aqualysin I with the C-terminal pro-sequence into the culture medium, and the precursor was then processed to the mature enzyme during the cultivation. However, the extracellular levels of aqualysin I in T. thermophilus cells harboring plasmids for deletion mutants as to the C-terminal pro-sequence were about 10–20% in comparison with the level of wild-type. Only the mature enzyme could be detected in the medium, while pro-aqualysin I with the C-terminal pro-sequence could not. These results suggest that the C-terminal pro-sequence of aqualysin I plays an important role in the extracellular secretion of aqualysin I.     

Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B

Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.     

Molecular dynamics as a tool to help design mutants

A 200 ps MD trajectory of wild type PCI and a 120 ps one for the Pro36Gly putative mutant are studied and compared with the structure of PCI in its complex with carboxypeptidase A (CPA). It is first established that the structures of PCI from X-ray and from MD simulation are essentially equal. Thereafter, data from the PCI-CPA and average MD structures together with available biochemical information are used to identify possible structural factors that may determine the inhibitory power of PCI. These structural determinants are used to analyze the mutant structure. The fold of the mutant protein shows a large degree of stability. The N-terminal tail in PCIm differs from the X-ray structure as it does in PCIw, while the mutant's C-terminal tail (which is the primary binding site with CPA) and residues 13–17 present deviations. Differences in fluctuation patterns exist between PCIm and PCIw in residues 2–4 (the N-terminal tail), 13–17, 22–23, 28–81 (the secondary contact site with CPA), and 37–38 (the C-terminal tail); the latter region is rigidified in PCIm. Results show that the MD method is able to sense long-range as well as local perturbative effects produced by amino-acid substitutions in flexible regions of this protein. The simulations suggest that the conformation of the C-terminal tail is less favorable for interaction with the target protein in the mutant than it is in the wild type protein. The Pro-36-Gly mutant is predicted to be a less potent inhibitor.Abbreviations CPA carboxypeptidase A - MD molecular dynamics - NIS non-inertial solvent - PCI potato carboxypeptidase A inhibitor - PCIm mutated inhibitor - PCIw wild inhibitor     

Characterization of the early steps of OE17 precursor transport by the thylakoid DeltapH/Tat machinery.

In order to probe the structure and protein translocation function of the thylakoid Tat machinery, a 25-residue C-terminal extension containing a 13-residue in vivo biotinylation tag and a 6x His tag was added to a mutant precursor of the 17-kDa subunit of the oxygen-evolving complex to form pOE17(C)-BioHis. When avidin was attached to biotinylated precursor in situ, the precursor-avidin complex was neither imported nor did it form a membrane-spanning translocation intermediate. It did, however, competitively inhibit the translocation of unbiotinylated precursor with an apparent KI unaffected by avidin. It is shown that the precursor protein achieves a stable folded structure upon dilution from urea, suggesting that the avidin-induced inhibition of transport results from a folding-induced proximity of N-terminal and C-terminal domains. It is further demonstrated that the majority of precursor rapidly binds to the thylakoid membrane, remaining import competent and yet undissociable by high salt or high pH treatment at ice temperature. The membrane binding event is unaffected by avidin. Import kinetics reveal that nonproton motive force-driven transport steps make up a major fraction of the transport time. These observations suggest that the N-terminal presequence on the avidin-bound precursor is available for membrane binding and initial recognition by the transport machinery, but the attached avidin signals the machinery that the precursor is an incorrectly configured substrate and thus import is aborted. Consequently, the DeltapH/Tat machinery's proofreading mechanism must operate after precursor recognition but before the committed step in transport.     

Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin.

The 220-kDa Bordetella pertussis filamentous hemagglutinin (FHA) is the major exported protein found in culture supernatants. The structural gene of FHA has a coding potential for a 367-kDa protein, and the mature form constitutes the N-terminal 60% of the 367-kDa precursor. The C-terminal domain of the precursor was found to be important for the high-level secretion of full-length FHA but not of truncated analogs (80 kDa or less). The secretion of full-length and truncated FHA polypeptides requires the presence of the approximately 100-amino-acid N-terminal domain and the outer membrane protein FhaC, homologous to the N-terminal domains of the Serratia marcescens and Proteus mirabilis hemolysins and their accessory proteins, respectively. By analogy to these hemolysins, it is likely that the N-terminal domain of the FHA precursor interacts, directly or indirectly, with the accessory protein during FHA biogenesis. However, immunogenicity and antigenicity studies suggest that the N-terminal domain of FHA is masked by its C-terminal domain and therefore should not be available for its interactions with FhaC. These observations suggest a model in which the C-terminal domain of the FHA precursor may play a role as an intramolecular chaperone to prevent premature folding of the protein. Both heparin binding and hemagglutination are expressed by the N-terminal half of FHA, indicating that this domain contains important functional regions of the molecule.