Role of the P6-P3' region of the serpin reactive loop in the formation and breakdown of the inhibitory complex.

Serpins have a large external peptide loop known as the reactive loop. Part of the reactive loop functions as the primary recognition site for target proteases; however, the complete role of the reactive loop in determining serpin specificity is unclear. In the current study, we investigated the reactive loop region that could potentially interact with the extended binding site of target proteases; the P6-P3' region. We utilized a reactive loop switching strategy to determine the extent to which the inhibitory activity of alpha-1-protease inhibitor (PI) against human neutrophil elastase (HNE) could be transferred to alpha-1-antichymotrypsin (ACT), a serpin that does not inhibit HNE. A series of ACT-PI chimeras were constructed in which segments of increasing length taken from the P6-P3' region of PI replaced the corresponding residues of ACT. The effectiveness of each chimera as an inhibitor of HNE was assessed by measuring (1) the rate of inhibitory complex formation and (2) the rate of complex breakdown (complex stability). Although all the ACT-PI chimeras were fully functional against chymotrypsin-like proteases, the series of chimeras showed no consistent progress toward the production of an inhibitor with the inhibitory properties of PI. The most rapid complex formation and most stable complexes were observed for chimeras with the P3-P1 residues of PI, whereas extending the replacement region to the P6 residue resulted in a considerable decrease in both inhibitory parameters. In order to study two additional features of the PI reactive loop that may play a role in the presentation of the P6-P3' region to HNE, we constructed variants that contained a P4' proline and deleted the P6'-P9' residues. Changes on the prime side appeared to have little effect on rates of inhibition or complex stability. Overall, even the most effective chimeras demonstrated an inhibition rate constant at least 60-fold less than that observed for PI inhibition of HNE and the most long lived chimera-HNE complexes broke down more rapidly than PI-HNE complexes. These results indicate that residues in the reactive loop region predicted to contact a specific target protease cannot fully transfer inhibitory activity from one serpin to another, suggesting that specific reactive loop-serpin body and serpin body-protease body interactions play a significant role in determining serpin inhibitory activity against target proteases.

Arginine substitutions in the hinge region of antichymotrypsin affect serpin beta-sheet rearrangement.

A hallmark of serpin function is the massive beta-sheet rearrangement involving the insertion of the cleaved reactive loop into beta-sheet A as strand s4A. This structural transition is required for inhibitory activity. Small hydrophobic residues at P14 and P12 positions of the reactive loop facilitate this transition, since these residues must pack in the hydrophobic core of the cleaved serpin. Despite the radical substitution of arginine at the P12 position, the crystal structure of cleaved A347R antichymotrypsin reveals full strand s4A insertion with normal beta-sheet A geometry; the R347 side chain is buried in the hydrophobic protein core. In contrast, the structure of cleaved P14 T345R antichymotrypsin reveals substantial yet incomplete strand s4A insertion, without burial of the R345 side chain.

Distortion of the active site of chymotrypsin complexed with a serpin.

There is no complete understanding of how serine protease inhibitors of the serpin family inhibit their target enzymes. Structural and biochemical studies have suggested that serpins utilize a mechanism that is distinct from the standard mechanism of inhibition proposed for most small protein protease inhibitors. Proton nuclear magnetic resonance spectroscopy was used in the present study to demonstrate a fundamental difference in the atomic environment of the catalytic triad of enzyme in complex with serpins when compared to uncomplexed enzyme and enzyme in complex with standard mechanism inhibitors. This work demonstrates that the active site of chymotrypsin is distorted when complexed to a serpin and makes tenable a mechanism of inhibition in which the serpin induces a conformational change in the enzyme that dramatically reduces or completely abrogates the catalytic activity of the protease.