Engineering thermostability in serine protease inhibitors

Abstract

Unlike most globular proteins, the native form of serine protease inhibitors (serpins) is strained. Previous studies of human α1- antitrypsin. a prototype plasma serpin, revealed that various unfavorable interactions, such as over-packing of side chains, buried polar groups and cavities, are the structural basis of the strain. The local strain could be relieved by various stabilizing single amino acid substitutions, which appeared to remove these unfavorable interactions. To improve the stability of other clinically important serpin members, here we examined whether the rules found in α1-antitrypsin studies are applicable to other serpins. Amino acid substitutions were introduced at various positions in human α1-antichymotrypsin and human antithrombin III that were equivalent to the sites of stabilizing substitutions of α1- antitrypsin. Two-thirds of the substitutions increased thermostability in all serpins tested. Mutational analysis and structural examination suggest that serpins are suboptimally folded using common structural strategies at many sites, even though some structural details can vary in individual members. The results suggest that schemes discovered with α1-antitrypsin, an easily manipulative serpin, are a useful basis for engineering conformational characteristics of other clinically important serpins.