ABSTRACT
The three-dimensional structures of engineered variants of Bacillus lentus subtilisin having increased enzymatic activity, K27R/N87S/V104Y/N123S/T274A (RSYSA) and N76D/N87S/S103A/V104I (DSAI), were determined by X-ray crystallography. In addition to identifying changes in atomic position we report a method that identifies protein segments having altered flexibility. The method utilizes a statistical analysis of variance to delineate main-chain temperature factors that represent significant departures from the overall variance between equivalent regions seen throughout the structure. This method reveals changes in main-chain mobility in both variants. Residues 125-127 have increased mobility in the RSYSA variant while residues 100-104 have decreased mobility in the DSAI variant. These segments are located at the substrate-binding site and changes in their mobility are believed to relate to the observed changes in proteolytic activity. The effect of altered crystal lattice contacts on segment flexibility becomes apparent when identical variants, determined in two crystal forms, are compared with the native enzyme.
Subject(s)
Bacillus/enzymology , Protein Engineering , Serine Endopeptidases/chemistry , Subtilisins/chemistry , Binding Sites , Crystallography, X-Ray , Isoenzymes/chemistry , Models, Molecular , Protein Conformation , Regression Analysis , Serine Endopeptidases/genetics , Subtilisins/genetics , TemperatureSubject(s)
Bacillus/enzymology , Genetic Variation , Mutagenesis, Site-Directed , Protein Engineering , Serine/chemistry , Subtilisins/chemistry , Bacillus/genetics , Computer Graphics , Hydrogen Bonding , Hydrolysis , Kinetics , Structure-Activity Relationship , Subtilisins/genetics , Subtilisins/physiologyABSTRACT
The crystallization of a variant of Bacillus lentus subtilisin and the native enzyme was achieved using identical conditions. The variant B. lentus was found to grow in two crystal forms, form 1 and form 2, whereas the native B. lentus subtilisin enzyme crystallized in only one, form 1. Form 2 crystals, once obtained, were found to grow much more rapidly than form 1 crystals. The lattice contacts and structural changes giving both crystal forms have been examined. The results show that crystal form 2 has a more complex network of interactions. There is also a small surface conformational change in the form 2 structure relative to the native and variant form 1 crystals and at least two solvent molecules bound to the enzyme in crystal form 1 are displaced in crystal form 2. In addition, a site specific substitution in the variant at position 27 induces a 'short' lattice contact which does not exist in the native B. lentus or the form 2 variant B. lentus. These results suggest that in some circumstances engineered variants could be designed to crystallize more rapidly than the native enzyme.
