The
serine protease inhibitor (
serpin) superfamily is involved in a wide range of cellular processes including fibrinolysis, angiogenesis, apoptosis,
inflammation,
metastasis and viral pathogenesis. Here, we investigate the unique mousetrap inhibition mechanism of
serpins through saturation mutagenesis of the P8 residue for a typical family member,
plasminogen activator inhibitor-2 (PAI-2). A number of studies have proposed an important role for the P8 residue in the efficient insertion and stabilisation of the cleaved reactive centre loop (RCL), which is a key event in the
serpin inhibitory mechanism. The importance of this residue for inhibition of the
PAI-2 protease target
urinary plasminogen activator (
urokinase, uPA) is confirmed, although a high degree of tolerance to P8 substitution is observed. Out of 19 possible
PAI-2 P8 mutants, 16 display inhibitory activities within an order of magnitude of the wild-type P8 Thr species. Crystal structures of complexes between
PAI-2 and RCL-mimicking
peptides with P8 Met or Asp mutations are determined, and structural comparison with the wild-type complex substantiates the ability of the S8 pocket to accommodate disparate side-chains. These data indicate that the identity of the P8 residue is not a determinant of efficient RCL insertion, and provide further evidence for functional plasticity of key residues within
enzyme structures. Poor correlation of observed
PAI-2 P8 mutant activities with a range of physicochemical, evolutionary and thermodynamic predictive indices highlights the practical limitations of existing approaches to predicting the molecular phenotype of
protein variants.