The role of strand 1 of the C beta-sheet in the structure and function of alpha(1)-antitrypsin

Abstract

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.

Fig. 1.

Structural diagram of the C β-sheet mutants of α₁-antitrypsin in comparison to the equivalent residues in antithrombin. (A) the structure of α₁-antitrypsin (pdb identifier 1QLP; Elliot et al. 1996 1998) with the C β-sheet mutations highlighted. The A β-sheet is in green, the B β-sheet in blue, the RCL in magenta, and strands s2C–s4C of the C β-sheet in yellow. Strand s1C is in aquamarine. P6′–P10′ are shown in red ball and stick. (B) Closeup of the C β-sheet region with the side chains of P6′–P10′ labeled. (C) Closeup on the C β-sheet region in antithrombin (pdb identifier 2ANT; Schreuder et al. 1994; Skinner et al. 1997) showing the hydrogen bond network between P10′R, P9′N, and 286P.

Fig. 2.

SDS-PAGE analysis of complex formation with thrombin by the S1C mutants of α₁-antitrypsin. α₁-AT was mixed with thrombin in a 2:1 molar ratio and allowed to incubate for 30 min at 25°C, following which the complexes were analyzed by 10% SDS-PAGE. The mutants analyzed are indicated, with P6′–P9′ mutants and wild type analyzed in (A), while the P10′ mutant is shown in (B). Incubation with thrombin (+) or not (−) is indicated . The positions of complexed, intact, and cleaved α₁-AT, as well as thrombin alone, are indicated. The molecular weight markers are shown in lanes marked "M", and their masses are indicated.

Fig. 3.

Thermal melts of two forms of α₁-AT indicating relative molecular stability. The P6′Ala (light gray line) and wild-type α₁-AT forms were analyzed by circular dichroism spectroscopy for their stability to thermal denaturation. Thermal unfolding was performed using a heating rate of 60°C/h, and the changes in secondary structure with temperature were measured by monitoring the CD signal at 230 nm.