Abstract
The subtilisin propeptide functions as an intramolecular chaperone (IMC) that 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. Existing data suggest that the chaperone and inhibitory functions of the subtilisin IMC domain are interdependent during folding. Based on x-ray structure of the IMC-subtilisin complex, we introduce a point mutation (E112A) to disrupt three hydrogen bonds that stabilize the interface between the protease and its IMC domain. This mutation within subtilisin does not alter the folding kinetics but dramatically slows down autoprocessing of the IMC domain. Inhibition of E112A-subtilisin activity by the IMC added in trans is 35-fold weaker than wild-type subtilisin. Although the IMC domain displays substantial loss of inhibitory function, its ability to chaperone E112A-subtilisin folding remains intact. Our results show that (i) the chaperone activity of the IMC domain is not obligatorily linked with its ability to bind with 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 for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin.
Highlights
The subtilisin propeptide functions as an intramolecular chaperone (IMC) that facilitates correct folding of the catalytic domain while acting like a competitive inhibitor of proteolytic activity
When the catalytic residue Ser221 is substituted with Cys, the precursor undergoes folding, autoprocessing, and structural changes subsequent to its autocleavage to give a complex in which the IMC domain remains associated with subtilisin through noncovalent interactions [31]
Based on the data presented in this manuscript both, IMC-subtilisin and IMC-E112A-subtilisin display similar folding rate constants (Table I)
Summary
The subtilisin propeptide functions as an intramolecular chaperone (IMC) that facilitates correct folding of the catalytic domain while acting like a competitive inhibitor of proteolytic activity. Our results show that (i) the chaperone activity of the IMC domain is not obligatorily linked with its ability to bind with and inhibit active subtilisin; (ii) degradation and not autoprocessing of the IMC domain is the rate-limiting step in precursor maturation; and (iii) the Glu112 residue within the IMCsubtilisin interface is not crucial for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin. Recent data show that the IMC domain can impart structural information onto its protease [27] and that this imprinting occurs after autoprocessing but before degradation of the IMC from the precursor [28] Such results suggest that IMCs promote protein folding by direct stabilization of the rate-limiting folding transition state and support the notion that the IMC is essential only during late stages of the folding process [29, 30]. It is important to note that the IMC domain and its fragments are devoid of secondary and tertiary structures when isolated from
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