Refined crystal structures of subtilisin novo in complex with wild-type and two mutant eglins. Comparison with other serine proteinase inhibitor complexes

J Mol Biol. 1991 Jan 20;217(2):353-71. doi: 10.1016/0022-2836(91)90549-l.


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.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Computer Graphics
  • Crystallography
  • DNA Mutational Analysis
  • Humans
  • Kinetics
  • Macromolecular Substances
  • Models, Molecular
  • Molecular Sequence Data
  • Motion
  • Pancreatic Elastase / antagonists & inhibitors
  • Protein Binding
  • Protein Conformation
  • Proteins
  • Recombinant Proteins / ultrastructure
  • Serine Proteinase Inhibitors* / pharmacology
  • Serpins*
  • Structure-Activity Relationship
  • Subtilisins / antagonists & inhibitors
  • Subtilisins / ultrastructure*
  • Trypsin Inhibitors


  • Macromolecular Substances
  • Proteins
  • Recombinant Proteins
  • Serine Proteinase Inhibitors
  • Serpins
  • Trypsin Inhibitors
  • eglin proteinase inhibitors
  • Subtilisins
  • Pancreatic Elastase