Inhibitory activity and conformational transition of α₁‐proteinase inhibitor variants

Abstract

Several variants of al-proteinase inhibitor (al-PJ) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) a,-Proteinase inhibitor (al-PI) is a member of the serine proteinase inhibitor (serpin) family [l -41. Inhibitors of this class play crucial roles in controlling proteolytic activities of major physiological importance, such as in coagulation, fibrinolysis and complement activation [5]. Several studies provided evidence that serpins are recognized by their target proteinases as substrates and that cleavage occurs between their P1 and PI' residues in the active site (positions 358 and 359 in al-PI) leading to a major conformational change in human al-P1 [6 -12al. More detailed information on how this conformational transition might take place was obtained from X-ray crystallographic analysis of the three-dimensional structure of al-PI cleaved at its active site by chymotrypsinogen. The most striking feature of this structure was that residues Met358 (Pl) and Ser359 (Pl') (see Fig. 1B) were separated by approximately 7 nm in the cleaved inhibitor [6]. It was hypothesized that insertion of residues 345 -358 of native a l -P1 into a pre-existing p-sheet in an antiparallel fashion was the key event of this conformational change. More recent studies on the structure of ovalbumin, a serpin with no inhibitory activity, and its cleaved form plakalbumin (see Fig. 1 A), which do not show this conformational change, confirmed this hypothesis and provided experimental evidence for the conformation of an uncleaved serpin [9, 131. In the follow- MATERIALS AND METHODS Production of a,-PI variantsOligonucleotide directed mutagenesis of the cDNA coding for [Met358 -+ Arg]al-PI with a deletion of its five N-termind amino acids was performed as previously described [15, 161. Expression of [Thr345 4 Arg, Met358 + ArgJal-Pi, [Met351 --$ Glu, Met358 -+Arg]a,-PI and [Met358 +Arg]al-PI was carried out in Escherichia coli (strain TGE 901) from plasmids pTG 7952, pTG 7955 and pTG 1943, respectively, under the control of the leftward promoter of phage 1 . in the presence of a temperature-sensitive repressor cl. For increased expression, a synthetic ribosome-binding site was employed [17]. Cultures were grown under selective conditions (200 mg/l ampicillin) at 30°C in a 20-1 fermenter (LSL Biolafitte, Saint-Germain en Laye, France) to an A600 of 10. Expression was induced by shifting the growth temperature to 42°C for 6 h. Isolation of the a,-PI variants was performed as previously described [18].

Characterization of a Human α1-Antitrypsin Variant That Is as Stable as Ovalbumin

“…For instance, the equilibrium unfolding transition of inhibitory serpins including ␣ 1 AT (Fig. 2) is very complex (5,(27)(28)(29), whereas the unfolding transition of ovalbumin is more or less fitted to a two-state model when monitored either by far UV CD signal (5), intrinsic fluorescence (30), or by UV absorbency and intrinsic viscosity (31). The unfolding midpoint of C73A mutant ovalbumin, which could not form disulfide bonds, was very close to that of Multi-7 ␣ 1 AT (Fig.…”

Structural Differences between Active Forms of Plasminogen Activator Inhibitor Type 1 Revealed by Conformationally Sensitive Ligands

“…We used a non-inhibitory PAI-1 mutant, PAI-1 R , which carries two arginyl residues at positions 333 and 335 in the proximal hinge, corresponding to RCL residues P12 and P14, but is otherwise identical to wtPAI-1 in primary amino acid sequence (23). Bulky charged residues at the proximal hinge have been shown to slow the insertion of the RCL into ␤-sheet A (45,46). Thus PAI-1 R behaves as a substrate and is completely cleaved by target proteases.…”

Structural aspects of serpin inhibition

“…Based on studies of naturally occurring mutants of the Cl inhibitor, Skriver et al [50] showed further effects also involving the P12 and PlO sites. Finally, since a major pathway for aggregation involves the complexation of strand s4A of one molecule in sheet A of another, aggregation studies also provide structural information [ 12, 15,48,49] and additionally demonstrate the mechanism of a type of serpin deficiency mutant syndrome.…”