Metastability in the inhibitory mechanism of human alpha1-antitrypsin

Metastability of the native form of proteins has been recognized as a mechanism of biological regulation. The energy-loaded structure of the fusion protein of influenza virus and the strained native structure of serpins (serine protease inhibitors) are typical examples. To understand the structural basis and functional role of the native metastability of inhibitory serpins, we characterized stabilizing mutations of alpha1-antitrypsin in a region presumably involved in complex formation with a target protease. We found various unfavorable interactions such as overpacking of side chains, polar-nonpolar interactions, and cavities as the structural basis of the native metastability. For several stabilizing mutations, there was a concomitant decrease in the inhibitory activity. Remarkably, some substitutions at Lys-335 increased the stability over 6 kcal mol-1 with simultaneous loss of activity over 30% toward porcine pancreatic elastase. Considering the location and energetic cost of Lys-335, we propose that this lysine plays a pivotal role in conformational switch during complex formation. Our current results are quite contradictory to those of previously reported hydrophobic core mutations, which increased the stability up to 9 kcal mol-1 without any significant loss of activity. It appears that the local strain of inhibitory serpins is critical for the inhibitory activity.