title: Refined crystal structures of subtilisin novo in complex with wild-type and two mutant eglins. Comparison with other serine proteinase inhibitor complexes.
abstract: The crystal structures of the complexes formed between subtilisin Novo and three inhibitors, eglin c, Arg45-eglin c and Lys53-eglin c have been determined using molecular replacement and difference Fourier techniques and refined at 2.4 A, 2.1 A, and 2.4 A resolution, respectively. The mutants Arg45-eglin c and Lys53-eglin c were constructed by site-directed mutagenesis in order to investigate the inhibitory specificity and stability of eglin c. Arg45-eglin became a potent trypsin inhibitor, in contrast to native eglin, which is an elastase inhibitor. This specificity change was rationalized by comparing the structures of Arg45-eglin and basic pancreatic trypsin inhibitor and their interactions with trypsin. The residue Arg53, which participates in a complex network of hydrogen bonds formed between the core and the binding loop of eglin c, was replaced with the shorter basic amino acid lysine in the mutant Lys53-eglin. Two hydrogen bonds with Thr44, located in the binding loop, can no longer be formed but are partially restored by a water molecule bound in the vicinity of Lys53. Eglin c in complexes with both subtilisin Novo and subtilisin Carlsberg was crystallized in two different space groups. Comparison of the complexes showed a rigid body rotation for the eglin c core of 11.5 degrees with respect to the enzyme, probably caused by different intermolecular contacts in both crystal forms.
authors.affiliation: Pharmaceutical Division, Ciba-Geigy Ltd., Basel, Switzerland.
authors.forename: D W
authors.initials: DW
authors.lastname: Heinz
authors.forename: J P
authors.initials: JP
authors.lastname: Priestle
authors.forename: J
authors.initials: J
authors.lastname: Rahuel
authors.forename: K S
authors.initials: KS
authors.lastname: Wilson
authors.forename: M G
authors.initials: MG
authors.lastname: Grütter
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003196:Computer Graphics; D003461:Crystallography; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins; D011994:Recombinant Proteins / Q000648:ultrastructure; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D015843:Serpins; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000648:ultrastructure*; D014361:Trypsin Inhibitors
chemical_list: D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D014361:Trypsin Inhibitors; C028656:eglin proteinase inhibitors; D013381:Subtilisins; D010196:Pancreatic Elastase