Identification of the interactions critical for propeptide-catalyzed folding of Tk-subtilisin.
Tk-subtilisin requires Ca(2+) for folding. This folding is accelerated by the chaperone function of its propeptide (Tkpro). Several Tkpro and Tk-subtilisin derivatives were constructed to examine whether the interactions between the C-terminal extended region of Tkpro and Tk-subtilisin and Glu61/Asp63- and Glu201-mediated hydrogen bonds at the domain interface are important for the chaperone function of Tkpro. The Tkpro derivatives with a series of C-terminal truncations and double mutations at Glu61 and Asp63 exhibited weaker chaperone functions than Tkpro for SA-subtilisin (active-site mutant of Tk-subtilisin). Good correlation was observed between their chaperone functions and binding abilities to the folded SA-subtilisin protein. These results suggest that the C-terminal extended region, Glu61, and Asp63 of Tkpro are not critical for folding of Tk-subtilisin but accelerate it by binding to a folding intermediate of Tk-subtilisin with a native-like structure at their binding sites. In contrast, Tkpro exhibited little chaperone function for E201A/SA-subtilisin. It could bind to the folded E201A/SA-subtilisin protein with a lower association constant than that for SA-subtilisin. These results suggest a loop of Tkpro, which interacts with Glu201 of Tk-subtilisin through hydrogen bonds and is required for folding of Tk-subtilisin by binding to a folding intermediate of Tk-subtilisin with a nonnative structure. Because this loop is fairly hydrophobic and tightly packs to the surface parallel helices of the central alphabetaalpha substructure of Tk-subtilisin, binding of this loop to Glu201 may induce association of these two helices and thereby formation of the alphabetaalpha substructure. We propose that Glu201-mediated interactions are critical for initiation of Tkpro-catalyzed folding of Tk-subtilisin.
Tanaka
Shun-ichi
S
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Matsumura
Hiroyoshi
H
Koga
Yuichi
Y
Takano
Kazufumi
K
Kanaya
Shigenori
S
D000595:Amino Acid Sequence; D002384:Catalysis; D002942:Circular Dichroism; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000201:enzymology*
D018832:Molecular Chaperones; D010455:Peptides; D018698:Glutamic Acid; D013381:Subtilisins