TY - JOUR
T1 - Folding pathway mediated by an intramolecular chaperone. The inhibitory and chaperone functions of the subtilisin propeptide are not obligatorily linked
AU - Fu, Xuan
AU - Inouye, Masayori
AU - Shinde, Ujwal
PY - 2000/6/2
Y1 - 2000/6/2
N2 - 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 Glu112 residue within the IMC-subtilisin interface is not crucial for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin.
AB - 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 Glu112 residue within the IMC-subtilisin interface is not crucial for initiating folding but is important in maintaining the IMC structure capable of binding subtilisin.
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U2 - 10.1074/jbc.275.22.16871
DO - 10.1074/jbc.275.22.16871
M3 - Article
C2 - 10828069
AN - SCOPUS:0034596066
SN - 0021-9258
VL - 275
SP - 16871
EP - 16878
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 22
ER -