Insights into the Serpin Inhibitory Mechanism from Structures of Mutant Serpins in the Mechaelis Complex

The serpins belong to a superfamily of protease inhibitors that employ a unique suicide substrate-like inhibitory mechanism. In this mechanism, target protease becomes acylated at the catalytic serine. Deacylation fails to take place, and the serpins undergo dramatic conformational changes in which the acylated protease is translocated 70Å from one pole of the serpin to the other. However, the factors causing suppression of deacylation are not fully understood. The previously solved Michaelis complex of serpin-1B with trypsin/S195A suggests that the P4 (I350) and P1' (S354) residues on the reactive center loop may be important factors in arresting the hydrolysis reaction, since these residues are involved in interactions with trypsin that are unique to serpins (unlike low molecular weight serine protease inhibitors). Inhibition assay data also show that the inhibitory activity of serpin was reduced by the introduction of a mutation either at the P4 (I350A) or at the P1' (S354A) position. The P4 (I350A) mutation nearly completely abolishes inhibitory activity of serpin-1B toward trypsin. In other words, the mutated P4 residue of serpin-1B acts as a substrate, rather than as an inhibitor. A crystallographic approach was used to understand why the serpin-1B (I350A) becomes a substrate; and, hopefully, to gain insight into the serpin inhibitory mechanism. Similarly, the formed Michaelis complex between trypsin and the serpin-1B (S354A) was also utilized to address the role of this residue in this serpin mechanism. The structural analysis shows that significant conformational changes were observed from the serpin-1B (I350A)/trypsin complex, but not from the serpin-1B (S354A)/trypsin complex. Due to these conformational changes, the special extensive interactions observed in the wild type complex were lost in the mutant complexes, the consequence being the destabilization of the mutant Michaelis complexes and, thus, perhaps destabilizing acylated covalent intermediate. Also, reduced interactions (mostly P-side of the serpin) induce a conformational change on trypsin Gln192, residue that may be important for arresting hydrolysis. Therefore, the stable and tight interactions in the Michaelis complex may be important in arresting deacylation in the serpin inhibitory mechanism.