Catalyis of a Protein Folding Reaction: Mechanistic Implications of the 2.0 Å Structure of the Subtilisin-Prodomain Complex

Philip Bryan, Lan Wang, Joel Hoskins, Sergei Ruvinov, Susan Strausberg, Patrick Alexander, Oma Almog, Gary Gilliland, Travis Gallagher

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121 Scopus citations


Biosynthesis of subtilisin is dependent on a 77 amino acid, N-terminal prodomain, which is autocatalytically processed to create the mature form of the enzyme [Ikemura, H., Takagi, H., & Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864], In order to better understand the role of the prodomain in subtilisin folding, we have determined the structure of the processed complex between the prodomain and subtilisin Sbt-70, a mutant engineered for facilitated folding. The prodomain is largely unstructured by itself but folds into a compact structure with a four-stranded antiparallel β-sheet and two three-turn a-helices when complexed with subtilisin. The Ka of the complex is 2 x 108 M-1 at 25 °C. The prodomain binds on subtilisin's two parallel surface a-helices and supplies caps to the N-termini of the two helices. The C-terminal strand of the prodomain binds in the subtilisin substrate binding cleft. While Sbt-70 is capable of independent folding, the prodomain accelerates the process by a factor of > 107 M-1 of prodomain in 30 mM Tris-HCl, pH 7.5, at 25 °C. X-ray structures of the mutant subtilisin folded in vitro either with or without the prodomain are compared and show that the identical folded state is acheived in either case. A model of the folding reaction of Sbt-70 and the prodomain is described as the following equilibria: P + Su ↔ Pf-SI ↔ Pf-Sf, where Su and P are Sbt-70 and prodomain, respectively, which are largely unstructured at the start of the reaction, Pf-SI is a collision complex of a partially folded Sbt-70 and folded prodomain, and Pf-Sf is the complex of folded Sbt-70 and prodomain. The mode of prodomain binding suggests that it catalyzes subtilisin folding by stabilizing the central aβa substructure in subtilisin. The prodomain bound to this substructure may correspond to the collision complex, Pf-SI, whose formation is rate limiting in the bimolecular reaction [Strausberg, S., Alexander, P., Wang, L., Schwarz, F., & Bryan, P. (1993) Biochemistry 32, 8112-8119].

Original languageEnglish
Pages (from-to)10310-10318
Number of pages9
Issue number32
StatePublished - 1 Jan 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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