Folding pathway mediated by an intramolecular chaperone: Dissecting conformational changes coincident with autoprocessing and the role of Ca2 in subtilisin maturation

Yukihiro Yabuta, Ezhilkani Subbian, Hiroshi Takagi, Ujwal Shinde, Masayori Inouye

Research output: Contribution to journalArticle

43 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)31-37
Number of pages7
JournalJournal of Biochemistry
Volume131
Issue number1
DOIs
StatePublished - Jan 1 2002

Keywords

  • Autoprocessing
  • Intramolecular chaperone
  • Propeptide
  • Protein folding
  • Subtilisin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Folding pathway mediated by an intramolecular chaperone: Dissecting conformational changes coincident with autoprocessing and the role of Ca<sup>2</sup> in subtilisin maturation'. Together they form a unique fingerprint.

  • Cite this