Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 A resolution.
Proteins of the subtilisin superfamily (subtilases) are widely distributed through many living species, where they perform a variety of processing functions. They are also used extensively in industry. In many of these enzymes, bound calcium ions play a key role in protecting against autolysis and thermal denaturation. We have determined the crystal structure of a highly thermostable protease from Bacillus sp. Ak.1 that is strongly stabilized by calcium. The crystal structure, determined at 1.8 A resolution (R=0. 182, Rfree=0.247), reveals the presence of four bound cations, three Ca(2+) and one Na(+). Two of the Ca(2+) binding sites, Ca-1 and Ca-2, correspond to sites also found in thermitase and the mesophilic subtilisins. The third calcium ion, however, is at a novel site that is created by two key amino acid substitutions near Ca-1, and has not been observed in any other subtilase. This site, acting cooperatively with Ca-1, appears to give substantially enhanced thermostability, compared with thermitase. Comparisons with the mesophilic subtilisins also point to the importance of aromatic clusters, reduced hydrophobic surface and constrained N and C termini in enhancing the thermostability of thermitase and Ak.1 protease. The Ak.1 protease also contains an unusual Cys-X-Cys disulfide bridge that modifies the active site cleft geometry.
Smith
C A
CA
School of Biological Sciences, University of Auckland, Auckland, New Zealand. ca.smith@auckland.ac.nz
Toogood
H S
HS
Baker
H M
HM
Daniel
R M
RM
Baker
E N
EN
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D011994:Recombinant Proteins; D013381:Subtilisins; D002118:Calcium