Catalysis of a protein folding reaction: mechanistic implications of the 2.0 A structure of the subtilisin-prodomain complex.
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 beta-sheet and two three-turn alpha-helices when complexed with subtilisin. The Ka of the complex is 2 x 10(8) M-1 at 25 degrees C. The prodomain binds on subtilisin's two parallel surface alpha-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 > 10(7) M-1 of prodomain in 30 mM Tris-HCl, pH 7.5, at 25 degrees 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 achieved 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Bryan
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P
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Wang
L
L
Hoskins
J
J
Ruvinov
S
S
Strausberg
S
S
Alexander
P
P
Almog
O
O
Gilliland
G
G
Gallagher
T
T
D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004792:Enzyme Precursors / Q000737:chemistry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013381:Subtilisins / Q000737:chemistry*
D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D013381:Subtilisins