Crystal structure of calcium-independent subtilisin BPN' with restored thermal stability folded without the prodomain

The three-dimensional struc- ture of a subtilisin BPN8 construct that was produced and folded without its prodomain shows the tertiary structure is nearly identical to the wild-type enzyme and not a folding interme- diate. The subtilisin BPN8 variant, Sbt70, was cloned and expressed in Escherichia coli without the prodomain, the 77-residue N-terminal do- main that catalyzes the folding of the enzyme into its native tertiary structure. Sbt70 has the high-affinity calcium-binding loop, residues 75 to 83, deleted. Such calcium-independent forms of subtilisin BPN8 refold independently while retaining high levels of activity (Bryan et al., Biochemistry, 31:4937-4945, 1992). Sbt70 has, in addition, seven stabilizing mutations, K43N, M50F, A73L, Q206V, Y217K, N218S, Q271E, and the active site serine has been replaced with alanine to prevent autolysis. The purified Sbt70 folded spontaneously without the prodomain and crystallized at room temperature. Crystals of Sbt70 belong to space group P212121 with unit cell parameters a 5 53.5 A, b 5 60.3 A, and c 5 83.4 A. Comparison of the refined structure with other high-resolution structures of subtili- sin BPN8 establishes that the conformation of Sbt70 is essentially the same as that previously determined for other calcium-independent forms and that of other wild-type subtilisin BPN8 structures, all folded in the presence of the prodomain. These findings confirm the re- sults of previous solution studies that showed subtilisin BPN8 can be refolded into a native conformation without the presence of the prodomain (Bryan et al., Biochemistry 31:4937- 4945, 1992). The structure analysis also pro- vides the first descriptions of four stabilizing mutations, K43N, A73L, Q206V, and Q271E, and provides details of the interaction between the enzyme and the Ala-Leu-Ala-Leu tetrapeptide found in the active-site cleft. Proteins 31:21-32, 1998. r 1998 Wiley-Liss, Inc.†