Tk-subtilisin requires Ca(2+) for folding. This folding is accelerated by the chaperone function of its propeptide (Tkpro). Several Tkpro and Tk-subtilisin derivatives were constructed to examine whether the interactions between the C-terminal extended region of Tkpro and Tk-subtilisin and Glu61/Asp63- and Glu201-mediated hydrogen bonds at the domain interface are important for the chaperone function of Tkpro. The Tkpro derivatives with a series of C-terminal truncations and double mutations at Glu61 and Asp63 exhibited weaker chaperone functions than Tkpro for SA-subtilisin (active-site mutant of Tk-subtilisin). Good correlation was observed between their chaperone functions and binding abilities to the folded SA-subtilisin protein. These results suggest that the C-terminal extended region, Glu61, and Asp63 of Tkpro are not critical for folding of Tk-subtilisin but accelerate it by binding to a folding intermediate of Tk-subtilisin with a native-like structure at their binding sites. In contrast, Tkpro exhibited little chaperone function for E201A/SA-subtilisin. It could bind to the folded E201A/SA-subtilisin protein with a lower association constant than that for SA-subtilisin. These results suggest a loop of Tkpro, which interacts with Glu201 of Tk-subtilisin through hydrogen bonds and is required for folding of Tk-subtilisin by binding to a folding intermediate of Tk-subtilisin with a nonnative structure. Because this loop is fairly hydrophobic and tightly packs to the surface parallel helices of the central alphabetaalpha substructure of Tk-subtilisin, binding of this loop to Glu201 may induce association of these two helices and thereby formation of the alphabetaalpha substructure. We propose that Glu201-mediated interactions are critical for initiation of Tkpro-catalyzed folding of Tk-subtilisin.