TY - JOUR
T1 - Folding pathway mediated by an intramolecular chaperone
T2 - Dissecting conformational changes coincident with autoprocessing and the role of Ca2 in subtilisin maturation
AU - Yabuta, Yukihiro
AU - Subbian, Ezhilkani
AU - Takagi, Hiroshi
AU - Shinde, Ujwal
AU - Inouye, Masayori
PY - 2002
Y1 - 2002
N2 - Subtilisin is produced as a precursor that requires its N-terminal propeptide to chaperone the folding of its protease domain. Once folded, subtilisin adopts a remarkably stable conformation, which has been attributed to a high affinity Ca2+ binding site. We investigated the role of the metal ligand in the maturation of pro-subtilisin, a process that involves folding, autoprocessing and partial degradation. Our results establish that although Ca2+ ions can stabilize the protease domain, the folding and autoprocessing of pro-subtilisin take place independent of Ca2+ ion. We demonstrate that the stabilizing effect of calcium is observed only after the completion of autoprocessing and that the metal ion appears to be responsible for shifting the folding equilibrium towards the native conformation in both mature subtilisin and the autoprocessed propeptide:subtilisin complex. Furthermore, the addition of active subtilisin to unautoprocessed pro-subtilisin in trans does not facilitate precursor maturation, but rather promotes rapid autodegradation. The primary cleavage site that initiates this autodegradation is at Gln19 in the N-terminus of mature subtilisin. This corresponds to the loop that links α-helix-2 and β-strand-1 in mature subtilisin and has indirect effects on the formation of the Ca2+ binding site. Our results show that the N-terminus of mature subtilisin undergoes rearrangement subsequent to propeptide autoprocessing. Since this structural change enhances the proteolytic stability of the precursor, our results suggest that the autoprocessing reaction must be completed before the release of active subtilisin in order to maximize folding efficiency.
AB - Subtilisin is produced as a precursor that requires its N-terminal propeptide to chaperone the folding of its protease domain. Once folded, subtilisin adopts a remarkably stable conformation, which has been attributed to a high affinity Ca2+ binding site. We investigated the role of the metal ligand in the maturation of pro-subtilisin, a process that involves folding, autoprocessing and partial degradation. Our results establish that although Ca2+ ions can stabilize the protease domain, the folding and autoprocessing of pro-subtilisin take place independent of Ca2+ ion. We demonstrate that the stabilizing effect of calcium is observed only after the completion of autoprocessing and that the metal ion appears to be responsible for shifting the folding equilibrium towards the native conformation in both mature subtilisin and the autoprocessed propeptide:subtilisin complex. Furthermore, the addition of active subtilisin to unautoprocessed pro-subtilisin in trans does not facilitate precursor maturation, but rather promotes rapid autodegradation. The primary cleavage site that initiates this autodegradation is at Gln19 in the N-terminus of mature subtilisin. This corresponds to the loop that links α-helix-2 and β-strand-1 in mature subtilisin and has indirect effects on the formation of the Ca2+ binding site. Our results show that the N-terminus of mature subtilisin undergoes rearrangement subsequent to propeptide autoprocessing. Since this structural change enhances the proteolytic stability of the precursor, our results suggest that the autoprocessing reaction must be completed before the release of active subtilisin in order to maximize folding efficiency.
KW - Autoprocessing
KW - Intramolecular chaperone
KW - Propeptide
KW - Protein folding
KW - Subtilisin
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U2 - 10.1093/oxfordjournals.jbchem.a003074
DO - 10.1093/oxfordjournals.jbchem.a003074
M3 - Article
C2 - 11754732
AN - SCOPUS:0036170846
SN - 0021-924X
VL - 131
SP - 31
EP - 37
JO - Journal of Biochemistry
JF - Journal of Biochemistry
IS - 1
ER -