CRYSTAL STRUCTURE OF SUBTILISIN BPN' FOLDED WITHOUT THE PRODOMAIN. Orna Almog, D. Travis Gallagher, Maria Tordova, Joel Hoskins, Phillip Bryan, Gary L. Gilliland. Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850

Secreted proteases such as subtilisin are usually synthesized as inactive proenzymes that are then processed into an active form. Once denatured, mature subtilisin BPN' refolds very slowly to the native state, but the refolding can be catalyzed by the addition of the prosegment [Bryan et al., (1995) Biochemistry 34, 10310-10318]. Deletion of the high-affinity calcium-binding site (A-site; residues 75-83) from subtilisin BPN' can accelerate the refolding of the mature protein to its native form. To help substantiate that refolding without the prodomain produces a native-like fold, Sbt70, a subtilisin BPN' variant, was cloned and expressed in an E. coli system without the prodomain. Sbt70 has, besides the deletion of the calcium binding site (residues 75-83), six stabilizing mutations, K43N, M50F, A73L, Q206V, Y217K and N218S, were introduced to restore lost thermal stability. The active-site serine was also replaced by alanine (S221A) to prevent autolysis. The isolated variant was folded without the prodomain by dialysis against phosphate buffer and crystallized. Crystals of Sbt70 belong to the space group P2₁2₁2₁ with unit cell parameters a=60.67 Å, b=84.02 Å, c=54.17 Å. The structure was solved by molecular replacement and refined using 2.2 _ data. The final R factor is 0.17. The structure comparison with other high-resolution subtilisin BPN' structures shows that the conformation of Sbt70 is virtually identical to that of natural mature subtilisin BPN'. An analysis of three previously unreported stabilizing mutations, K43N, A73L, and Q206V, will also be described along with the interactions with the enzyme of the tetra-peptide ALAL that was unexpectedly found bound in the active site.