A Pathway for Conformational Diversity in Proteins Mediated by Intramolecular Chaperones

Abstract

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.

Folding Pathway Mediated by an Intramolecular Chaperone

“…The predicted structure in boldface represents predictions that do not match with the secondary structure obtained from the crystallized complex of ProWT⅐subtilisin. Motifs N1 and N2 represent the conserved domains within the subtilase family (18). Asterisks denote residues that constitute the hydrophobic core within the propeptide.…”

“…Because propeptides perform functions different from the catalytic domains, they may be subjected to different mutational frequencies because of different functional constraints (14 -16). However, given that they impart structural information to their catalytic domains (17)(18)(19), propeptides within one family could adopt similar structural scaffolds despite digressions in their polypeptide sequences. This makes propeptides attractive models for protein redesign and for understanding the relation between sequence and structure.…”

Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence

“…A prosequence of subtilisins has been shown to function not only as an intramolecular chaperone but also as a template for molecular imprinting that facilitates correct folding of the catalytic domain (29,30). This prosequence should be removed from the catalytic domain by autoprocessing or by another protease upon completion of the protein folding (31).…”

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.…”