Folding pathway mediated by an intramolecular chaperone - The inhibitory and chaperone functions of the subtilisin propeptide are not obligatorily linked

The subtilisin propeptide functions as an intramolecular chaperone (IMC) th at facilitates correct folding of the catalytic domain while acting like a competitive inhibitor of proteolytic activity. Upon completion of folding, subtilisin initiates IMC degradation to complete precursor maturation. Exis ting data suggest that the chaperone and inhibitory functions of the subtil isin IMC domain are interdependent during folding. Based on x-ray structure of the IMC-subtilisin complex, we introduce a point mutation (E112A) to di srupt three hydrogen bonds that stabilize the interface between the proteas e and its IMC domain. This mutation within subtilisin does not alter the fo lding kinetics but dramatically slows down autoprocessing of the IMC domain . Inhibition of E112A-subtilisin activity by the IMC added in trans is 35-f old weaker than wild-type subtilisin. Although the IMC domain displays subs tantial loss of inhibitory function, its ability to chaperone E112A-subtili sin folding remains intact. Our results show that (i) the chaperone activit y of the IMC domain is not obligatorily linked with its ability to bind wit h and inhibit active subtilisin; (ii) degradation and not autoprocessing of the IMC domain is the rate-limiting step in precursor maturation; and (iii ) the Glu(112) residue within the IMC-subtilisin interface is not crucial f or initiating folding but is important in maintaining the IMC structure cap able of binding subtilisin.