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 propeptid e (ProA) to function as an intramolecular chaperone (IMC). To decipher the mechanisms through which propeptides can initiate protein folding, we chara cterized ProA in terms of its sequence, structure and function. Our results show that, in contrast to ProS (propeptide of subtilisin), ProA can fold s pontaneously, reversibly and cooperatively into a stable monomeric alpha-be ta 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-globu le folding intermediate. Moreover, despite showing only 21% sequence identi ty with ProS, ProA can not only inhibit enzymatic activity with a magnitude tenfold greater than ProS, but can also chaperone subtilisin folding, albe it with a lower efficiency. The structure of ProA complexed with subtilisin is different from that of isolated ProA. Hence, additional interactions se em necessary to induce ProA into a compact structure. Our results also sugg est that: (a) propeptides that are potent inhibitors are not necessarily be tter IMCs; (b) propeptides within the subtilase family appear polymorphic a nd; (c) the intrinsic instability within propeptides may be necessary for r apid activation of the cognate protein. (C) 2001 Academic Press.