Folding Pathway Mediated by an Intramolecular Chaperone

Self Cite

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 wildtype 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 ␣؊␤ conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT⅐subtilisin. 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.Subtilisin E is an alkaline serine protease isolated from Bacillus subtilis (1). In vivo this protein is produced as pre-prosubtilisin (2), wherein the pre-region (signal peptide) facilitates protein secretion and the pro-region (propeptide) functions as an intramolecular chaperone that guides correct folding of subtilisin (2-6). Upon completion of folding, the propeptide is autoproteolytically removed (7). This is necessary because the propeptide is a potent inhibitor of subtilisin activity (4).Propeptide-mediated folding mechanisms exist in several unrelated protease families. However, there is no sequence conservation in propeptide domains within these families (8). This functional conservation across unrelated protease families suggests that propeptides have evolved in multiple parallel pathways and may share a common mechanism of action (9). Subtilisin (2-6) and ␣-lytic protease (10 -13) constitute the best-studied examples of propeptide-mediated protein folding. Bacterial subtilisins are models for the subtilase family that spans prokaryotes and eukaryotes. Amino acid sequence analysis of this family establishes that alth...

Section: Discussionsupporting

confidence: 54%

“…Cells were grown in M9 medium supplemented with 50 g/ml ampicillin (22). At an absorbance of 0.6 A 600 nm , the culture was rapidly cooled to 12°C.…”

Section: Methodsmentioning

confidence: 99%

Folding Pathway Mediated by an Intramolecular Chaperone

Yabuta

Subbian

Oiry

et al. 2003

Self Cite

“…The fact that the BACE Pro region promotes folding but is not a potent inhibitor of the protease is unusual but not without precedent. A recent study showed that the inhibitory and chaperone-like functions of the subtilisin Pro peptide are not obligatorily linked (26). It should be noted, however, that additional studies are required to exclude the possibility that membrane-tethered, mammalian-cell derived, full-length ProBACE is a strict zymogen with respect to its ability to process its natural membrane-bound substrate, APP.…”

Section: Discussionmentioning

confidence: 99%

The Pro Domain of β-Secretase Does Not Confer Strict Zymogen-like Properties but Does Assist Proper Folding of the Protease Domain

Shi¹,

Chen²,

Yin³

et al. 2001

122

␤-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid ␤ peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4 Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k cat /K m of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4 Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/ renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

“…Studies are in progress to determine whether the N-terminal propeptide of SspB has retained the function of an intramolecular chaperone to facilitate folding of SspB or whether this function is also satisfied by SspC. Others have shown that the chaperone and inhibitor functions of the subtilisin propeptide are not obligatorily linked to one another, such that mutations that eliminate inhibitory activity do not interfere with chaperone function (53). Therefore, it is possible that the N-terminal propeptide of pSspB retains a function as an intramolecular chaperone.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Maturation Mechanism and Restricted Substrate Specificity for the SspB Cysteine Protease ofStaphylococcus aureus

Massimi¹,

Park²,

Rice³

et al. 2002

106

The SspB cysteine protease of Staphylococcus aureus is expressed in an operon, flanked by the sspA serine protease, and sspC, encoding a 12.9-kDa protein of unknown function. SspB was expressed as a 40-kDa prepropeptide pSspB, which did not undergo autocatalytic maturation. Activity of pSspB was reduced compared with 22-kDa mature SspB, but it was equivalent to mature SspB after incubation with SspA, which specifically removed the pSspB N-terminal propeptide. SspC abrogated the activity of pSspB when incubated in a 1:1 complex but had no effect on SspA or papain. Activity of the pSspB⅐SspC complex was restored when incubated with SspA, and SspC was cleaved by SspA but not pSspB. Thus, SspC maintains pSspB as an inert zymogen, and SspA is required for removal of the propeptide and inactivation of SspC. Like the papain protease family, SspB cleaved substrates with a hydrophobic amino acid at P2 but had a strong preference for arginine at P1. It did not cleave casein, serum albumin, IgG, or IgA, but it promoted detachment of cultured keratinocytes and cleaved fibronectin and fibrinogen at sites recognized by urokinase plasminogen activator and plasmin, respectively. It also processed high molecular weight kininogen in a manner resembling plasma kallikrein. Thus, SspB exhibits a novel maturation mechanism and mimics the specificity of plasma serine proteases.