Calcium-mediated thermostability in the subtilisin superfamily: The crystal structure of Bacillus Ak.1 protease at 1.8 angstrom resolution

Proteins of the subtilisin superfamily (subtilases) are widely distributed through many Living species, where they perform a variety of processing fun ctions. They are also used extensively in industry. In many of these enzyme s, bound calcium ions play a key role in protecting against autolysis and t hermal denaturation. We have determined the crystal structure of a highly t hermostable protease from Bacillus sp. Ak.1 that is strongly stabilized by calcium. The crystal structure, determined at 1.8 Angstrom resolution (R=0. 182, R-free = 0.247), reveals the presence of four bound cations, three Ca2 + and one Naf. Two of the Ca2+ binding sites, Ca-l and Ca-2, correspond to sites also found in thermitase and the mesophilic subtilisins. The third ca lcium ion, however, is at a novel site that is created by two key amino aci d substitutions near Ca-l, and has not been observed in any other subtilase . This site, acting cooperatively with Ca-l, appears to give substantially enhanced thermostability, compared with thermitase. Comparisons with the me sophilic subtilisins also point to the importance of aromatic clusters, red uced hydrophobic surface and constrained N and C termini in enhancing the t hermostability of thermitase and Ak.1 protease. The Ak.1 protease also cont ains an unusual Cys-X-Cys disulfide bridge that modifies the active site cl eft geometry. (C) 1999 Academic Press.