The role of strand 1 of the C β‐sheet in the structure and function of α₁‐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 ␤-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 ␣ 1 -antitrypsin Pittsburgh mutant (P1 ‫ס‬ Arg). Mutation of the P10Ј position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7Ј and P9Ј caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6Ј and P8Ј positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6Ј and P8Ј residues of the C ␤-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, P6Ј and P8Ј mutants of ␣ 1 -antitrypsin had melting temperatures 14 degrees lower than wild-type ␣ 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.

The appended tail region of heparin cofactor II and additional reactive centre loop mutations combine to increase the reactivity and specificity of α1-proteinase inhibitor M358R for thrombin

“…We first confirmed thatthe RCL5 loop behavedsimilarlyin our α 1 -PIM358R expression system as previouslyreported (19). Reported ratec onstants for recombinant α 1 -PIM 358R inhibition of thrombin vary,evenbetween laboratoriesemploying bacterial expression (from 6 [38] to 11 [20] to 29 X10 6 M -1 min -1 [32]). Similarly, Hopkinsetal.…”

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

“…We have previously reported that API made in this soluble, intracellular bacterial expression system, exhibits SI values of 2–3 [29] , [30] , [41] . Others employing different bacterial expression systems, some in which recombinant API was renatured from inclusion bodies, have reported SI values closer to unity [55] , [56] . Why the difference arises is not fully understood, but we have demonstrated functionality of API M358R made in this system as an antithrombotic agent in vivo [39] , suggesting that it does not have an unnatural fold.…”

α1‐Proteinase inhibitor mutants with specificity for plasma kallikrein and C1s but not C1

“…Changing Glu to Ala probably alters the orientation of the residue side chain away from the solvent towards the body of the protein, which in turn causes loss of stabilizing interactions such as the hydrogen bond. The integrity of β‐sheet C (of which this region is strand 1), is likely to be critical for the correct folding of the serpin in the native metastable state (Eldering et al 1995; Patston and Gettins 1996; Chang et al 1997; Bottomley et al 2001). Another mutant in which we changed the P9′ residue of the α 1 ‐PI‐ LGR Gln to Glu also formed polymers, consistent with this idea (data not shown).…”