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Thermitase is a thermostable member of the subtilisin family of serine
proteases. Four independently determined crystal structures of the enzyme are
compared in this study: a high resolution native one and three medium resolution
complexes of thermitase with eglin-c, grown from three different calcium
concentrations. It appeared that the B-factors of the thermitase eglin complex
obtained at 100 mM CaCl2 and elucidated at 2.0 A resolution are remarkably
similar to those of the 1.4 A native structure: the main chain atoms have an rms
difference of only 2.3 A2; for all atoms this difference is 4.6 A2. The rms
positional differences between these two structures of thermitase are 0.31 A for
the main chain atoms and 0.58 A for all atoms. There results show that not only
atomic positions but also temperature factors can agree well in X-ray structures
determined entirely independently by procedures which differ in virtually every
possible technical aspect. A detailed comparison focussed on the effects of
eglin binding on the structure of thermitase. Thermitase can be considered as
consisting of (1) a central core of 94 residues, plus (2) four segments of 72
residues in total which shift as rigid bodies with respect to the core, plus (3)
the remaining 113 residues which show small changes but, however, cannot be
described as rigid bodies. The central cores of native thermitase and the 100 mM
CaCl2 thermitase:eglin complex have an rms deviation of 0.13 A for 376 main
chain atoms. One of the segments, formed by loops of the strong calcium binding
site, shows differences up to 1.0 A in C alpha positions. These are probably due
to crystal packing effects. The three other segments, comprising 51 residues,
are affected conformational changes upon eglin binding so that the P1 to P3
binding pockets of thermitase broaden by 0.4 to 0.7 A. The residues involved in
these changes correspond with residues which change position upon inhibitor
binding in other subtilisins. This suggests that an induced fit mechanism is
operational during substrate recognition by subtilisins.
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