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

Erythrocyte invasion by the malaria merozoite is prevented by serine protease inhibitors. Various aspects of the biology of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1), including the timing of its expression and its apical location in the merozoite, suggest that this enzyme is involved in invasion. Recombinant PfSUB-1 expressed in a baculovirus system is secreted in the p54 form, noncovalently bound to its cognate propeptide, p31. To understand the role of p31 in PfSUB-1 maturation, we examined interactions between p31 and both recombinant and native enzymes. CD analyses revealed that recombinant p31 (rp31) possesses significant secondary structure on its own, comparable with that of folded propeptides of some bacterial subtilisins. Kinetic studies demonstrated that rp31 is a fast binding, high affinity inhibitor of PfSUB-1. Inhibition of two bacterial subtilisins by rp31 was much less effective, with inhibition constants 49 -60-fold higher than that for PfSUB-1. Single (at the P4 or P1 position) or double (at P4 and P1 positions) point mutations of residues within the C-terminal region of rp31 had little effect on its inhibitory activity, and truncation of 11 residues from the rp31 C terminus substantially reduced, but did not abolish, inhibition. None of these modifications prevented binding to the PfSUB-1 catalytic domain or rendered the propeptide susceptible to proteolytic digestion by PfSUB-1. These studies provide new insights into the function of the propeptide in PfSUB-1 activation and shed light on the structural requirements for interaction with the catalytic domain.Parasites of the genus Plasmodium are responsible for the debilitating disease malaria, which affects 300 -500 million people each year, mostly in subtropical regions (1). The continued emergence of drug-resistant parasites imposes an urgent need for a new generation of control measures. The life cycle of the malaria parasite in the human is complex, involving intracellular replication in both hepatocytes and erythrocytes, with the pathophysiological manifestations closely allied to intraerythrocytic replication. Plasmodium merozoites actively penetrate erythrocytes via a tightly regulated series of events involving proteolytic processing and release of proteins from the apical organelles and surface of the parasite (2-4). Functional maturation of many invasion-related proteins requires their proteolytic modification and, in some cases, their eventual removal from the parasite surface. These processing events can occur both in the apical organelles and at the parasite surface (4 -6), suggesting a role for proteases in different compartments of the parasite. Specific inhibitors of cysteine and serine proteases can block invasion, providing compelling evidence that these classes of parasite proteases play a major role in the invasion process (6, 7). In the case of Plasmodium falciparum, the species responsible for most fatal cases of malaria, two subtilisin-like serine proteases (subtilases) have been identified, called PfSUB-...

Section: Discussionmentioning

confidence: 99%

Functional Characterization of the Propeptide of Plasmodium falciparum Subtilisin-like Protease-1

Jean¹,

Hackett²,

Martin³

et al. 2003

“…This is necessary because the propeptide can also inhibit subtilisin activity (3). Enzyme activity was measured using N-suc-AAPF-pNA as a synthetic substrate (23). TPCK-treated trypsin does not cleave the synthetic substrate and hence does not interfere with the activity assay.…”

Section: Methodsmentioning

confidence: 99%

“…2C) and represent averages of three independent scans. For the fluorescence measurements, the denatured and folded ProD (7.0 M) were excited at 295 nm, and the emission spectra from 310 to 410 nm were recorded as described earlier (23).…”

Section: Methodsmentioning

confidence: 99%

Folding Pathway Mediated by an Intramolecular Chaperone

Yabuta

Subbian

Oiry

et al. 2003

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...

“…A growing family of membrane and secretory proteins contain pro-domains at the N-terminal end that undergo post-translational proteolytic cleavage in the late secretory pathway after the protein has acquired transport competence (26,(33)(34)(35)(36). These domains, called intramolecular chaperones, modulate the folding of this class of proteins directly through interaction with other regions of the protein or by substituting for ER resident chaperones.…”

Section: Expression Of a Full-length Im-mentioning

confidence: 99%

Sucrase Is an Intramolecular Chaperone Located at the C-terminal End of the Sucrase-Isomaltase Enzyme Complex

Jacob¹,

Pürschel²,

Naim³

2002

The sucrase-isomaltase enzyme complex (pro-SI) is a type II integral membrane glycoprotein of the intestinal brush border membrane. Its synthesis commences with the isomaltase (IM) subunit and ends with sucrase (SUC). Both domains reveal striking structural similarities, suggesting a pseudo-dimeric assembly of a correctly folded and an enzymatically active pro-SI. The impact of each domain on the folding and function of pro-SI has been analyzed by individual expression and coexpression of the individual subunits. SUC acquires correct folding, enzymatic activity and transport competence and is secreted into the external milieu independent of the presence of IM. By contrast, IM persists as a mannose-rich polypeptide that interacts with the endoplasmic reticulum resident molecular chaperone calnexin. This interaction is disrupted when SUC is coexpressed with IM, indicating that SUC competes with calnexin for binding of IM. The interaction between SUC and the membrane-anchored IM leads to maturation of IM and blocks the secretion of SUC into the external milieu. We conclude that SUC plays a role as an intramolecular chaperone in the context of the pro-SI protein.To our knowledge all intramolecular chaperones so far identified are located at the N-terminal end. SUC is therefore the first C-terminally located intramolecular chaperone in mammalian cells.Membrane and secretory proteins are subject to a complex array of cotranslational and post-translational processes that precede sorting to the organelle in which they exert their biological functions. The most crucial events take place in the ER 1 and include folding, subunit assembly, and in many cases oligomerization (for review see Ref. 1). These steps, individually or altogether, lead ultimately to the acquisition of the protein to a transport-competent conformation that enables its egress from the ER. A number of resident proteins of the ER, such as the molecular chaperones BiP and calnexin and protein disulfide isomerase, play primordial roles in these processes that vary from binding of unfolded proteins to the formation of disulfide bonds between individual subunits or within a single polypeptide chain (2-4). A large number of proteins assemble into homodimers, oligomers, or heterodimers preceding protein exit from the ER (5, 6). Correct protein folding of the individual monomers or subunits is required for optimal completion of these events. Many proteins are known not to go into dimers or heterodimers, such as the brush border proteins sucrase-isomaltase (SI) (7), angiotensin-converting enzyme (8), and maltase-glucoamylase (9). An interesting and common feature of these proteins is their composition of an even number of homologous domains. SI, for example, is an enzyme complex that is made of two subunits that share Ն 35% of amino acid sequence similarity and 41% of conserved sequences (10). Although no three-dimensional structure of this large protein is so far available, algorithmic predictions suggest that the two large domains are folded similarly and that...