title: A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases.
abstract: Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability. The predominant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal inactivation. Thermal inactivation is not caused by the absence of calcium from the Ca3 site per se, but rather by unfolding of a region of TLP-ste for which stability depends on the occupancy of the Ca3 site. In accordance with this concept is the observation that the effects of mutations in the Ca3 site could be compensated by stabilizing mutations near this site. In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely stable TLP-ste variants containing multiple stabilizing mutations in the Ca3 region. Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivation.
authors.affiliation: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
authors.forename: O R
authors.initials: OR
authors.lastname: Veltman
authors.forename: G
authors.initials: G
authors.lastname: Vriend
authors.forename: H J
authors.initials: HJ
authors.lastname: Berendsen
authors.forename: B
authors.initials: B
authors.lastname: Van den Burg
authors.forename: G
authors.initials: G
authors.lastname: Venema
authors.forename: V G
authors.initials: VG
authors.lastname: Eijsink
mesh_terms: D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006358:Hot Temperature; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; D002118:Calcium