The native strains in the hydrophobic core and flexible reactive loop of a serine protease inhibitor : crystal structure of an uncleaved α₁-antitrypsin at 2.7 A

Abstract

Background: The protein α1-antitrypsin is a prototype member of the serpin (serine protease inhibitor) family and is known to inhibit the activity of neutrophil elastase in the lower respiratory tract. Members of this family undergo a large structural rearrangement upon binding to a target protease, involving cleavage of the reactive-site loop. This loop is then inserted into the main body of the enzyme following the opening of a central β sheet, leading to stabilization of the structure. Random mutageneses of α1-antitrypsin identified various mutations that stabilize the native structure and retard the insertion of the reactive-site loop. Structural studies of these mutations may reveal the mechanism of the conformational change. Results: We have determined the three-dimensional structure of an uncleaved α1-antitrypsin with seven such stabilizing mutations (hepta α1- antitrypsin) at 2.7 A resolution. From the comparison of the structure with other serpin structures, we found that hepta α1-antitrypsin is stabilized due to the release of various strains that exist in native wild type α1- antitrypsin, including unfavorable hydrophobic interactions in the central hydrophobic core. The reactive-site loop of hepta α1-antitrypsin is an extended strand, different from that of the previously determined structure of another uncleaved α1-antitrypsin, and indicates the inherent flexibility of the loop. Conclusions: The present structural study suggests that the uncleaved α1-antitrypsin has many folding defects which can be improved by mutations. These folding defects seem to be utilized in a coordinated fashion in the regulation of the conformational switch of α1-antitrypsin. Some of the defects, represented by the Phe51 region and possibly the Met374 and the Thr59 regions, are part of the sheet-opening mechanism.