Folding Pathway Mediated by an Intramolecular Chaperone

Abstract

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...

Insights from Bacterial Subtilases into the Mechanisms of Intramolecular Chaperone-Mediated Activation of Furin

“…Alignment of known IMC sequences from subtilases helped identify two small hydrophobic motifs, N1 and N2, that appear to be conserved within such propeptides (70). Interestingly, when one compares the IMC sequence from subtilisin E with aqualysin, POIA1 (71), or with a designed peptide chaperone ProD which was computationally forced to diverge from the IMC of subtilisin, a high degree of sequence conservation appears isolated within motifs N1 and N2 (72). Random mutagenesis using error-prone PCR along with an activity-based genetic screening technique demonstrated that substitutions within motifs N1 and N2 were often deleterious (73, 74).…”

“…To explore this possibility, the sequence–structure relation within subtilases was analyzed (72). It is easier to reconcile the differences in the extent of sequence conservation among the IMCs and their cognate protease domains in different protein families.…”

“…Not surprisingly, the chaperoning ability and inhibitory functions of IMC domains correlate well, and IMC variants with diminished binding affinity are often weaker chaperones (73, 79, 80). However, this correlation is not always true, as the propeptide of aqualysin (a closely related thermostable homologue of subtilisin) (81) and a computationally designed synthetic peptide are potent slow-binding inhibitors (72), yet much weaker chaperones of subtilisin. An examination of other convergently evolved IMC-dependent protease systems establishes that the slow-binding inhibition function of IMC domains appears to be a common theme within proteinase A, α-lytic protease, carboxypeptidase Y, elastase, and the cathepsin family.…”

CK2 Phosphorylates SSRP1 and Inhibits Its DNA-binding Activity

“…CD analyses were performed on an automated AVIV 215 spectrometer at 25 °C as described previously (27). All the protein concentrations were maintained at 0.1 mg/ml.…”

Identification of Interaction Site of Propeptide toward Mature Carboxypeptidase Y (mCPY) Based on the Similarity between Propeptide and CPY Inhibitor (IC)

“…16,17) In a study of serine protease subtilisins and their propeptides, the functions of subtilisin propeptides as intramolecular chaperons and inhibitors were identified, [18][19][20] and the structure of the subtilisinpropeptide complex was also determined. 20) Structural analysis indicated that the C-terminal strand of the propeptide binds to the subtilisin substrate-binding cleft.…”