Conformation of the reactive site loop of .alpha.1-proteinase inhibitor probed by limited proteolysis

Abstract

Elucidation of the reactive site loop (RSL) structure of serpins is essential for understanding their inhibitory mechanism. Maintenance of the RSL structure is likely to depend on its interactions with a dominant unit of secondary structure known as the A-sheet. We investigated these interactions by subjecting alpha 1-proteinase inhibitor to limited proteolysis using several enzymes. The P1-P10 region of the RSL was extremely sensitive to proteolysis, indicating that residues P3'-P13 are exposed in the virgin inhibitor. Following cleavage eight or nine residues upstream from the reactive site, the protein noncovalently polymerized, sometimes forming circles. Polymerization resulted from insertion of the P1-P8 or P1-P9 region of one molecule into the A-sheet of an adjacent proteolytically modified molecule. The site of cleavage within the RSL had a distinct effect on the conformational stability of the protein, such that stability increased as more amino acids insert into the A-sheet. We conclude that the A-sheet of virgin alpha 1-proteinase inhibitor resembles that of ovalbumin, except that it contains a bulge where two or three RSL residues are inserted. Insertion of seven or eight RSL residues, allowed by proteolytic cleavage of the RSL, causes expansion of the sheet. It is likely that the RSL of alpha 1-proteinase inhibitor and several serpins exhibits significantly more mobility than is common among other protein inhibitors of serine proteinases.

Interaction of subtilisins with serpins

“…Therefore, the temperature dependence of the partition ratio reflects the temperature dependence of the inhibitory activity of alP1 and aIACT. Beyond 45 "C, ( Y~P I starts to polymerize as visualized previously on native-PAGE (Mast et al, 1992), and the inhibitory activity drops.…”

“…The precipitate was air dried, and subjected to SDS-PAGE analysis after boiling for 5 min in sample buffer containing 1 Yo SDS and 20 mM DTT. TUG gels were cast with a 0-8 M urea gradient in 7% acrylamide gels as previously described (Goldenberg, 1989;Mast et al, 1992).…”

Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of α1-antitrypsin

“…1G) were prepared by incubating plasma M ␣ 1 -antitrypsin at 1 mg/mL with 0.01 mg/mL of papaya proteinase IV (PP4; CN Biosciences, Nottingham, UK), a glycine-specific cysteine protease that cleaves ␣ 1 -antitrypsin at the P9-P10 bond of the reactive loop, at 37°C for 2 hours. 28 Recombinant Glu342Ala and His334Ala ␣ 1 -antitrypsin were expressed and purified as described previously. 29 Polymers of these mutants were prepared by heating at 0.5 mg/mL and 50°C (His334Ala ␣ 1 -antitrypsin) or 55°C (Glu342Ala Western Blot Analysis.…”

Inhibition of coagulation factors by recombinant barley serpin BSZx

“…BSZx retained nearly full activity after preincubation with 8E8 in excess and, presumably, the epitope recognized by 8E8 is not located in the loop region. More likely, partial or complete loop insertion caused by complex formation or cleavage results in a structural rearrangement disrupting the epitope [19][20][21]. This suggestion is Min Fig.…”