CRYSTAL-STRUCTURE OF CALCIUM-INDEPENDENT SUBTILISIN BPN' WITH RESTORED THERMAL-STABILITY FOLDED WITHOUT THE PRODOMAIN

The three-dimensional structure of a subtilisin BPN' construct that wa s produced and folded without its prodomain shows the tertiary structu re is nearly identical to the wild-type enzyme and not a folding inter mediate. The subtilisin BPN' variant, Sbt70, was cloned and expressed in Escherichia coli without the prodomain, the 77-residue N-terminal d omain that catalyzes the folding of the enzyme into its native tertiar y structure. Sbt70 has the high-affinity calcium-binding loop, residue s 75 to 83, deleted. Such calcium-independent forms of subtilisin BPN' refold independently while retaining high levels of activity [Bryan e t al., Biochemistry, 31:4937-4945, 1992]. Sbt70 has, in addition, seve n 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 prodoma in and crystallized at room temperature. Crystals of Sbt70 belong to s pace group P2(1)2(1)2(1) with unit cell parameters a = 53.5 Angstrom, b = 60.3 Angstrom, and c = 83.4 Angstrom. Comparison of the refined st ructure with other high-resolution structures of subtilisin BPN' estab lishes 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 BPN' structures, all folded in the presence of the prodomain. These findings confirm the results of previous solu tion studies that showed subtilisin BPN' can be refolded into a native conformation without the presence of the prodomain [Bryan et al., Bio chemistry 31:4937-4945, 1992]. The structure analysis also provides th e 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. (C) 1998 Wiley-Liss, Inc.