Propeptide Of Subtilisin Research Articles

Four residues of propeptide are essential for precursor folding of nattokinase.

Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding. Single-site and multi-site mutagenesis studies confirmed the role of Tyr10, Gly13, Gly34, and Gly35. During stage (i) and (ii) of precursor folding, Tyr10 and Gly13 would form the part of interface with subtilisin domain. While Gly34 and Gly35 connected with an α-helix that would stabilize the structure of propeptide. The quadruple Ala mutation, Y10A/G13A/G34A/G35A, resulted in a loss of the chaperone function for the propeptide. This work showed the essential residues of propeptide for precursor folding via secondary structure and kinetic parameter analyses.

Mycobacterium tuberculosis MycP1 Protease Plays a Dual Role in Regulation of ESX-1 Secretion and Virulence

Mycobacterium tuberculosis uses the ESX-1 secretion system to deliver virulence proteins during infection of host cells. Here we report a mechanism of posttranscriptional control of ESX-1 mediated by MycP1, a M. tuberculosis serine protease. We show that MycP1 is required for ESX-1 secretion but that, unexpectedly, genetic inactivation of MycP1 protease activity increases secretion of ESX-1 substrates. We demonstrate that EspB, an ESX-1 substrate required for secretion, is a target of MycP1 in vitro and in vivo. During macrophage infection, an inactive MycP1 protease mutant causes hyperactivation of ESX-1-stimulated innate signaling pathways. MycP1 is required for growth in mice during acute infection, while loss of its protease activity leads to attenuated virulence during chronic infection. As the key ESX-1 substrates ESAT-6 and CFP-10 are highly immunogenic, fine-tuning of their secretion by MycP1 may balance virulence and immune detection and be essential for successful maintenance of long-term M. tuberculosis infection.

Inhibitor-assisted refolding of protease: A protease inhibitor as an intramolecular chaperone

Pleurotus ostrearus proteinase A inhibitor 1 (POIA1), which was discovered as a protease inhibitor, is unique in that it shows sequence homology to the propeptide of subtilisin, which functions as an intramolecular of a cognate protease. In this study, we demonstrate that POIA1 can function as an intramolecular chaperone of subtilisin by in vitro and in vivo experiments. The specific cleavage between POIA1 and the mature region of subtilisin BPN′ occurred in a refolding process of a chimera protein, and Bacillus cells transformed with a chimera gene formed a halo on a skim milk plate. The mutational analyses of POIA1 in the chimera protein suggested that the tertiary structure of POIA1 is required for such a function, and that an increase in its ability to bind to subtilisin BPN′ makes POIA1 a more effective intramolecular chaperone.

Folding Pathway Mediated by an Intramolecular Chaperone: A FUNCTIONAL PEPTIDE CHAPERONE DESIGNED USING SEQUENCE DATABASES

Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or "intramolecular chaperones" to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined alpha-beta conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT.subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.

Structure of POIA1, a homologous protein to the propeptide of subtilisin: implication for protein foldability and the function as an intramolecular chaperone

Solution structure of POIA1 ( Pleurotus ostreatus proteinase A inhibitor 1), which functions as an intramolecular chaperone and as an inhibitor to subtilisin, was determined. By making use of the fact that POIA1 is the only structured protein that shows homology to the propeptide of subtilisin, which is unstructured by itself, foldability of this protein was elucidated. It became clear that the evolutionarily conserved residues play two important roles, one for the maintenance of its own structure, and the other for the interaction with subtilisin. Structural softness and mutational tolerance contained in the POIA1 structure makes it an ideal material for designing a foldable protein.

Alteration of Inhibitory Properties of Pleurotus ostreatus Proteinase A Inhibitor 1 by Mutation of Its C-Terminal Region

Pleurotus ostreatus proteinase A inhibitor 1 (POIA1), which is composed of 76 residues without disulfide bridges, is a unique inhibitor in that it exhibits sequence similarity to the propeptides of subtilisins. In order to elucidate the inhibitory mechanism of POIA1, we constructed an expression system for a synthetic POIA1 gene. The wild-type POIA1 was found to inhibit subtilisin BPN′ with an inhibitor constant (Ki) of 3.2 × 10−9 M, but exhibited a time-dependent decrease of inhibitory activity as a consequence of degradation by the protease, showing that the wild-type POIA1 was a temporary inhibitor when subtilisin BPN′ was used as a target protease. Since POIA1 shows sequence similarity to the propeptide of subtilisin, which is known to inhibit the protease via its C-terminal region, the C-terminal six residues of POIA1 were replaced with those of the propeptide of subtilisin BPN′. The mutated POIA1 inhibited subtilisin BPN′ with a Ki value of 2.8 × 10−11 M and did not exhibit time-dependent decrease of inhibitory activity, showing about 100-fold increases in binding affinity for, and resistance to, the protease. These results clearly indicate that the C-terminal region of POIA1 plays an important role in determining the inhibitory activity toward the protease, and that the increase in binding ability to the protease is closely related to resistance to proteolytic degradation. Therefore, the inhibitory properties of POIA1 can be altered by mutation of its C-terminal region.

Backbone dynamics of the natively unfolded pro-peptide of subtilisin by heteronuclear NMR relaxation studies.

The dynamics of the natively unfolded form of the pro-peptide of subtilisin (PPS) have been characterized at two different pHs (6.0 and 3.0) by 15N relaxation experiments. 15N relaxation data is obtained at multiple field strengths and a detailed comparison of spectral density mapping, the model free approach and the recently proposed Cole-Cole model free (CC-MF) analysis is presented. The CC-MF analysis provides a better fit to the observed magnetic field dependence of 15N relaxation data of unfolded PPS than conventional model free approaches and shows that fluctuations in R2 may be accounted for by a distribution of correlation times on the nanosecond timescale. A new parameter epsilon derives from the analysis and represents the width of the distribution function and the heterogeneity of the dynamics on the nanosecond timescale at a particular site. Particularly interesting is the observation that epsilon is sensitive to pH changes and that PPS samples a wider distribution of nanosecond time scale motions at less acidic pHs than at more acidic pHs. These results suggest that PPS experiences a higher degree of correlated motion at pH 6.0 and that electrostatic interactions may be important for inducing correlated motions on the nanosecond timescale in unfolded PPS.

Folding pathway mediated by an intramolecular chaperone: the structural and functional characterization of the aqualysin I propeptide

Aqualysin I, a thermostable homologue of subtilisin, requires its propeptide (ProA) to function as an intramolecular chaperone (IMC). To decipher the mechanisms through which propeptides can initiate protein folding, we characterized ProA in terms of its sequence, structure and function. Our results show that, in contrast to ProS (propeptide of subtilisin), ProA can fold spontaneously, reversibly and cooperatively into a stable monomeric α-β conformation, even when isolated from its cognate protease-domain. ProA displays an indiscernible amount of tertiary structure with a considerable solvent-accessible hydrophobic surface, but is not a classical molten-globule folding intermediate. Moreover, despite showing only 21 % sequence identity with ProS, ProA can not only inhibit enzymatic activity with a magnitude tenfold greater than ProS, but can also chaperone subtilisin folding, albeit with a lower efficiency. The structure of ProA complexed with subtilisin is different from that of isolated ProA. Hence, additional interactions seem necessary to induce ProA into a compact structure. Our results also suggest that: (a) propeptides that are potent inhibitors are not necessarily better IMCs; (b) propeptides within the subtilase family appear polymorphic and; (c) the intrinsic instability within propeptides may be necessary for rapid activation of the cognate protein.

Folding Pathway Mediated by an Intramolecular Chaperone: THE INHIBITORY AND CHAPERONE FUNCTIONS OF THE SUBTILISIN PROPEPTIDE ARE NOT OBLIGATORILY LINKED

The subtilisin propeptide functions as an intramolecular chaperone (IMC) that facilitates correct folding of the catalytic domain while acting like a competitive inhibitor of proteolytic activity. Upon completion of folding, subtilisin initiates IMC degradation to complete precursor maturation. Existing data suggest that the chaperone and inhibitory functions of the subtilisin IMC domain are interdependent during folding. Based on x-ray structure of the IMC-subtilisin complex, we introduce a point mutation (E112A) to disrupt three hydrogen bonds that stabilize the interface between the protease and its IMC domain. This mutation within subtilisin does not alter the folding kinetics but dramatically slows down autoprocessing of the IMC domain. Inhibition of E112A-subtilisin activity by the IMC added in trans is 35-fold weaker than wild-type subtilisin. Although the IMC domain displays substantial loss of inhibitory function, its ability to chaperone E112A-subtilisin folding remains intact. Our results show that (i) the chaperone activity of the IMC domain is not obligatorily linked with its ability to bind with and inhibit active subtilisin; (ii) degradation and not autoprocessing of the IMC domain is the rate-limiting step in precursor maturation; and (iii) the Glu(112) residue within the IMC-subtilisin interface is not crucial for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin.

A Pathway for Conformational Diversity in Proteins Mediated by Intramolecular Chaperones

Conformational diversity within unique amino acid sequences is observed in diseases like scrapie and Alzheimer's disease. The molecular basis of such diversity is unknown. Similar phenomena occur in subtilisin, a serine protease homologous with eukaryotic pro-hormone convertases. The subtilisin propeptide functions as an intramolecular chaperone (IMC) that imparts steric information during folding but is not required for enzymatic activity. Point mutations within IMCs alter folding, resulting in structural conformers that specifically interact with their cognate IMCs in a process termed "protein memory." Here, we show a mechanism that mediates conformational diversity in subtilisin. During maturation, while the IMC is autocleaved and subsequently degraded by the active site of subtilisin, enzymatic properties of this site differ significantly before and after cleavage. Although subtilisin folded by Ile-48 --> Thr IMC (IMCI-48T) acquires an "altered" enzymatically active conformation (SubI-48T) significantly different from wild-type subtilisin (SubWT), both precursors undergo autocleavage at similar rates. IMC cleavage initiates conformational changes during which the IMC continues its chaperoning function subsequent to its cleavage from subtilisin. Structural imprinting resulting in conformational diversity originates during this reorganization stage and is a late folding event catalyzed by autocleavage of the IMC.

Involvement of the C-terminal region of yeast proteinase B inhibitor 2 in its inhibitory action

Yeast proteinase B inhibitor 2 (YIB2), which is composed of 74 amino acid residues, is an unusual serine protease inhibitor, since it lacks disulfide bonds. To identify its reactive site for proteases, we constructed an expression system for a synthetic YIB2 gene and then attempted to change the inhibitory properties of YIB2 by amino acid replacements. The purified wild-type YIB2 inhibited the activity of subtilisin BPN′, a protein homologous to yeast proteinase B, although its binding ability was not strong, and a time-dependent decrease in its inhibitory activity was observed, demonstrating that wild-type YIB2 behaves as a temporary inhibitor when subtilisin BPN′ is the target protease. Since YIB2 exhibits sequence homology to the propeptide of subtilisin, which inhibits a cognate protease using its C-terminal region, we replaced the six C-termi nal residues of YIB2 with those of the propeptide of subtilisin BPN′ to make the mutant YIB2m1. This mutant exhibited markedly increased inhibitory activity toward subtilisin BPN′ without a time-dependent decrease in its inhibitory activity. Replacement of only the C-terminal Asn of YIB2 by Tyr, or deletion of the C-terminal Tyr of YIB2m1, inhibited subtilisin, but the ability of these mutants to bind subtilisin and their resistance to proteolytic attack were weaker than those of YIB2m1, indicating that the C-terminal residue contributes to the interaction with the protease to a greater extent than the preceding five residues and that the resistance of YIB2 to proteolyic attack is closely related to its ability to bind a protease. These results demonstrate that YIB2 is a unique protease inhibitor that involves its C-terminal region in the interaction with the protease.

Protein memory through altered folding mediated by intramolecular chaperones.

The 77-residue propeptide of subtilisin acts as an intramolecular chaperone that organizes the correct folding of its own protease domain. Similar folding mechanisms are used by several prokaryotic and eukaryotic proteins, including prohormone-convertases. Here we show that the intramolecular chaperone of subtilisin facilitates folding by acting as a template for its protease domain, although it does not form part of that domain. Subtilisin E folded by an intramolecular chaperone with an Ile(-48)-to-Val mutation acquires an 'altered' enzymatically active conformation that differs from wild-type subtilisin E. Although both the altered and wild-type subtilisins have identical amino-acid sequences, as determined by amino-terminal sequencing and mass spectrometry, they bind their cognate intramolecular chaperones with 4.5-fold greater affinity than non-cognate intramolecular chaperones, when added in trans. The two subtilisins also have different secondary structures, thermostability and substrate specificities. Our results indicate that an identical polypeptide can fold into an altered conformation through a mutated intramolecular chaperone and maintains memory of the folding process. Such a phenomenon, which we term 'protein memory', may be important in investigations of protein folding.

Ab initio structure prediction for small polypeptides and protein fragments using genetic algorithms.

Ab initio folding simulations have been performed on three peptides, using a genetic algorithm-based search method which operates on a full atom representation. Conformations are evaluated with an empirical force field parameterized by a potential of mean force analysis of experimental structures. The dominant terms in the force field are local and nonlocal main chain electrostatics and the hydrophobic effect. Two of the simulated structures were for fragments of complete proteins (eosinophil-derived neurotoxin (EDN) and the subtilisin propeptide) that were identified as being likely initiation sites for folding. The experimental structure of one of these (EDN) was subsequently found to be consistent with that prediction (using local hydrophobic burial as the determinant for independent folding). The simulations of the structures of these two peptides were only partly successful. The most successful folding simulation was that of a 22-residue peptide corresponding to the membrane binding domain of blood coagulation factor VIII (Membind). Three simulations were performed on this peptide and the lowest energy conformation was found to be the most similar to the experimental structure. The conformation of this peptide was determined with a C alpha rms deviation of 4.4 A. Although these simulations were partly successful there are still many unresolved problems, which we expect to be able to address in the next structure prediction experiment.

Functional Analysis of the Propeptide of Subtilisin E as an Intramolecular Chaperone for Protein Folding: REFOLDING AND INHIBITORY ABILITIES OF PROPEPTIDE MUTANTS

The amino-terminal propeptide, consisting of 77 amino acid residues, is known to be required as an intramolecular chaperone to guide the folding of mature subtilisin E, a serine protease, into active mature enzyme. Many mutations within the pro-sequence have been shown to abolish the production of active subtilisin E (Kobayashi, T., and Inouye, M. (1992) J. Mol. Biol. 226, 931-933). Here we report characterization, refolding, and inhibitory abilities of six single amino acid substitution mutations (Ile-67-->Val, Ile-48-->Thr, Gly-44-->Asp, Lys-36-->Glu, Ala-30-->Thr, and Pro-15-->Leu) and a nonsense mutation (N59-mer) at the codon for Lys-18. These mutant propeptides were expressed in Escherichia coli using a T7 expression system and were purified to homogeneity. Surprisingly, Lys-36-->Glu, Ala-30-->Thr and Pro-15-->Leu were found to still function as a chaperone for in vitro refolding of denatured subtilisin BPN' with 60, 80, and 54% efficiency compared to the wild-type propeptide, respectively. The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively. The Ki values against subtilisin BPN' were 1.6 x 10(-9) M, and 2.1 x 10(-9) M, respectively, almost identical to the Ki value exhibited by the wild-type propeptide (1.4 x 10(-9) M). In contrast, Ile-67-->Val and Gly-44-->Asp were able to refold denatured subtilisin BPN' with only 18 and13% efficiencies and had Ki values of 10 and 11 x 10(-9) M, respectively. The Ile-48-->Thr mutant propeptide was unable to refold denatured subtilisin BPN' and gave a 100-fold higher Ki (118 x 10(-9) M) than the wild-type propeptide. The N59-mer propeptide extending from Leu-19 to Met-78 was unable to function as a chaperone. Like the wild-type propeptide, none of the mutant propeptides had secondary structures as judged by their circular dichroism spectra. The present results demonstrate that the ability of the propeptide as a chaperone to refold the denatured protein is well correlated with its ability as a competitive inhibitor for the active enzyme. This supports the notion that the secondary and tertiary structures of the propeptide are identical or highly homologous between the renatured propeptide-subtilisin complex and the inhibitory complex formed between the propeptide and the active enzyme.

A covalently trapped folding intermediate of subtilisin E: spontaneous dimerization of a prosubtilisin E Ser49Cys mutant in vivo and its autoprocessing in vitro.

The propeptide of subtilisin E (the N-terminal 77 amino acid extension) is required for the proper folding of the nascent mature protein and is also a potent and specific inhibitor of the active enzyme. Previous studies have demonstrated that the propeptide can renature denatured mature sequence either in cis or in trans and can be considered an intramolecular chaperone, since it is not required for activity of the mature enzyme. In this paper it is shown that a prosubtilisin-S49C mutant can be expressed in Escherichia coli either as a monomer or as a disulfide-linked dimer, (prosubtilisin-S49C)2, depending on the vector selected. Interconversion between (prosubtilisin-S49C)2 and prosubtilisin-S49C could be readily achieved by reduction and oxidation in denaturing solutions, such as guanidine hydrochloride or urea. While the monomer can undergo autoprocessing in vitro under refolding conditions, the dimer is trapped in an intermediate state which could not be processed into active enzyme. Remarkably, the autoprocessing of this trapped intermediate could be induced readily upon reduction by dithiothreitol. This disulfide-linked (prosubtilisin-S49C)2 is fairly stable, but does tend to aggregate when the ionic strength of the solution is reduced below 0.1 M. The disulfide-linked (prosubtilisin-S49C)2 has far- and near-UV CD spectra revealing the presence of both secondary and tertiary structures, respectively, similar to those of the active mature monomer. Hence this autoprocessing-competent state appears to be a "late" folding intermediate, arising after the "molten globule" state formed in the absence of the prosequence, that has no discernible tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Folding pathway mediated by an intramolecular chaperone.

The N-terminal propeptide of subtilisin, a serine protease, functions as an intramolecular chaperone which is crucial for proper folding of the active enzyme. This nascent N-terminal propeptide is removed after completion of the folding process. Here we present a possible pathway by which intramolecular chaperones mediate protein folding. Using circular dichroism to analyze acid-denatured subtilisin we have identified a folding-competent state which can refold to an active conformation in the absence of the propeptide. Earlier work had shown that guanidine hydrochloride-denatured subtilisin was in a state incapable of folding in absence of its propeptide. Comparison of the folding-incompetent and folding-competent states indicates that refolding is facilitated by the presence of residual structure present only in the folding-competent state. The analysis further indicates that the propeptide is essential for inducing this state. Therefore the folding-competent state may lie on--or be in rapid equilibrium with an intermediate on--the folding pathway of subtilisin. In the absence of the propeptide, formation of such a state--and hence refolding--is extremely slow.

Catalysis of a protein folding reaction: thermodynamic and kinetic analysis of subtilisin BPN' interactions with its propeptide fragment.

The in vivo folding of subtilisin is dependent on a 77 amino acid propeptide, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme (Ikemura et al., 1987). We have cloned, expressed, and purified large quantities of the 77 amino acid subtilisin propeptide. This has enabled us to characterize its participation in the subtilisin folding reaction by spectroscopic and microcalorimetric methods. Unfolded subtilisin, when returned to native conditions, is kinetically isolated from its native state. Folding of subtilisin with the native calcium site-A is extremely slow even in the presence of a high concentration of isolated propeptide. The folding of a calcium-free mutant subtilisin, however, is readily catalyzed by the isolated propeptide. The propeptide-subtilisin folding reaction can be described as the following equilibrium: P(u) + S(u)<==>P-S<==>Pf-Sf<==>P(u) + Sf, where S(u) and P(u) are subtilisin and propeptide, respectively, which are largely unstructured at the start of the reaction; P-S is a collision complex of unfolded subtilisin and propeptide; Pf-Sf is the complex of folded subtilisin and propeptide; and Sf is folded subtilisin. The rate-limiting step in the folding reaction of calcium-free mutant subtilisin is formation of the initial collision complex, P-S. The rate at which P(u) and S(u) form a productive collision complex is approximately 500 M-1 s-1. The collision complex appears to be an early folding unit which, once formed, results in rapid isomerization to the fully folded complex. The rate constant for isomerization of the collision complex to the folded complex is > or = 0.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional analysis of the intramolecular chaperone: Mutational hot spots in the subtilisin pro-peptide and a secondsite suppressor mutation within the subtilisin molecule

The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.

Intramolecular Chaperone: The Role of the Pro-Peptide in Protein Folding

Subtilisin, an alkaline serine protease, is produced in the bacterium as pre-pro-subtilisin; the pre-peptide of 29 amino acid residues is the signal peptide essential for the secretion of prosubtilisin from the cytoplasm into the culture medium. On the other hand, the pro-peptide of 77 residues covalently linked to the amino terminal end of the subtilisin intramolecularly guides the folding of subtilisin into the active enzyme. Importantly, the pro-peptide is not required for the enzymatic activity and is removed intramolecularly by autoprocessing upon the completion of the protein folding. In this review, I will first summarize all the data concerning the functions of the subtilisin pro-peptide. On the basis of these results, I shall discuss a new general concept, an intramolecular chaperone to explain the essential role of the pro-peptide in protein folding.