The native strains in the hydrophobic core and flexible reactive loop of a serine protease inhibitor: Crystal structure of an uncleaved alpha(1)-antitrypsin at 2.7 angstrom

Background: The protein alpha(1)-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 beta sheet, leading to stabilization of the structure. Random mutageneses of alpha(1)-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 alpha(1)-antitrypsin with seven such stabilizing mutations (hepta alpha(1)-antitrypsin) at 2.7 Angstrom resolution. From the comparison of the structure with other serpin structures, we found that hepta alpha(1)-antitrypsin is stabilized due to the release of various strains that exist in native wild type alpha(1)-antitrypsin, including unfavorable hydrophobic interactions in the central hydrophobic core. The reactive-site loop of hepta alpha(1)-antitrypsin is an extended strand, different from that of the previously determined structure of another uncleaved alpha(1)-antitrypsin, and indicates the inherent flexibility of the loop. Conclusions: The present structural study suggests that the uncleaved alpha(1)-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 alpha(1)-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.