Subtilases and metal binding – the weak binding site of subtilisins revisited
J. Dohnálek1, K. MacAuley2, A. M. Brzozowski2, T. V. Borchert3, K. S. Wilson2
1Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 16206 Praha 6, Czech Republic
2Structural Biology Laboratory, University of York, Heslington, York, YO10 5YW, United Kingdom
3Novozymes A/S, Brudelysvej 26, DK-2880 Bagsværd, Denmark
Subtilisins form a family of a/b hydrolases with broad specificity towards the cleaved peptide sequence . Relationship between their structure and activity was extensively studied by means of x-ray crystallography. Representatives of this family such as subtilisin BPN´/NOVO, Carlsberg or Savinase became subjects of systematic studies of metal ion binding in relation to the enzymatic activity, stability and structure since 1969 when the first subtilisin structure was published . Enzymes from the superfamily of subtilisin-like proteases have their catalytic domain homologous to that of subtilisins with some additional domains and/or secondary structure elements.
In the earlier studies a strong calcium binding site (CaI, formerly named site A) and a weak calcium binding site (CaII, formerly named site B) were identified in subtilisins . The site CaI plays a crucial role in stabilizing the molecular structure but the role of the site CaII is slightly more complicated and depends on concentration of sodium ions in the environment . As the number of structures of subtilisins in the PDB  was growing it became obvious that in some cases the contents of site CaII was misinterpreted.
Two novel structures of subtilisin-like proteases were determined by single crystal x-ray diffraction. The extracellular proteases are produced by bacteria Bacillus sp. TY145 and Bacillus halmapalus. The new subtilases bring experimental evidence for Na+ instead of Ca2+ bound in site NaII (2.7 Å away from site CaII) and for three new Ca2+-binding sites in subtilases. We have attempted to re-classify the typical metal binding sites of subtilisins and subtilisin-like proteases and to reflect on the most likely nature of the ions in site CaII in the publicly available structures.
This work was supported by Novozymes, Denmark and by the Ministry of Education, Youth and Sport of the Czech Republic (project no. 1K05008).