Tertiary structure formation in the propeptide of subtilisin BPN' by successive amino acid replacements and its close relation to function.

The propeptide of subtilisin BPN', located between a signal peptide and the mature region of the protease, is known to exhibit inhibitory activity toward subtilisin BPN', in addition to its activity as an intramolecular chaperone that facilitates folding of subtilisin BPN'. Another unique feature is that although the isolated propeptide is in a random-coil state, it forms a defined tertiary structure when it is bound to subtilisin BPN'. In this study, amino acid replacements likely to increase the hydrophobicity of the propeptide have been introduced so that the isolated propeptide forms a defined tertiary structure. By successive replacements of Ala47 by Phe, Gly13 by Ile and Val65 by Ile, the propeptide was found to form a tertiary structure in addition to an increase in its secondary structure content, which were identified by circular dichoism spectra measurements. Concurrently, the propeptide, which is a temporary inhibitor in its wild-type form, became resistant to proteolytic digestion by subtilisin BPN'. These results show not only the close relationship between tertiary structure formation in the propeptide and its function as a protease inhibitor but also the ability of a random-coil protein to form a tertiary structure after a limited number of well-designed amino acid replacements. Copyright 1998 Academic Press Limited.