Cavities of α 1 ‐antitrypsin that play structural and functional roles

The native form of inhibitory serine protease inhibitors (serpins) is strained, which is critical for their inhibitory activity. Previous studies on stabilizing mutations of α 1 ‐antitrypsin, a prototype of serpins, indicated that cavities provide a structural basis for the native strain of the molecule. We have systematically mapped the cavities of α 1 ‐antitrypsin that play such structural and functional roles by designing cavity‐filling mutations at residues that line the walls of the cavities. Results show that energetically unfavorable cavities are distributed throughout the α 1 ‐antitrypsin molecule, and the cavity‐filling mutations stabilized the native conformation at 8 out of 10 target sites. The stabilization effect of the individual cavity‐filling mutations of α 1 ‐antitrypsin varied (0.2–1.9 kcal/mol for each additional methylene group) and appeared to depend largely on the structural flexibility of the cavity environment. Cavity‐filling mutations that decreased inhibitory activity of α 1 ‐antitrypsin were localized in the loop regions that interact with β‐sheet A distal from the reactive center loop. The results are consistent with the notion that β‐sheet A and the structure around it mobilize when α 1 ‐antitrypsin forms a complex with a target protease.