Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A .beta.-sheet of the inhibitor during trapping of target proteinases

Abstract

Identical or highly similar antigenic determinants, not present in the intact inhibitor, were induced in antithrombin on cleavage of the reactive bond, on formation of a complex between antithrombin and a synthetic reactive-loop tetradecapeptide, and on partial denaturation of antithrombin at low concentrations of guanidinium chloride. Previous studies indicate that the common structural feature of these three modified forms of antithrombin is that the region of the reactive-bond loop on the amino-terminal side of the reactive bond, or the corresponding synthetic peptide, is inserted as a middle strand in the main beta-sheet of the inhibitor, the A sheet. The new epitopes in the three modified antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed also in complexes between antithrombin and thrombin or factor Xa, strongly suggesting that a substantial segment of the reactive-bond loop is inserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, implying that the conformational change induced by heparin does not involve such loop insertion. These results provide the first experimental verification of recent hypotheses that insertion of the reactive-bond loop of serpins into the A beta-sheet is involved in the binding of target proteinases.

Identification of the Antithrombin III Heparin Binding Site

“…Comparison of the P14-inserted ATIII structure with those of cleaved bovine ATIII and latent human ATIII (data not shown) indicates that the configuration of heparin binding site residues changes upon deeper insertion of the reactive loop polypeptide into sheet A, as would occur during inhibitory complex formation and cleavage of the reactive loop. These changes in geometry are suggested to decrease the affinity of the inhibitory complex and cleaved ATIII for heparin and may reflect the overall protein conformational change that also causes certain epitope(s) to be absent from native and heparin bound ATIII, but exposed in inhibitory complexes of ATIII with thrombin and factor Xa, and in cleaved ATIII and binary complexes of ATIII with a reactive loop peptide (6). Consistent with decreased heparin affinity following inhibitory complex formation or ATIII cleavage, deeper insertion of the reactive loop polypeptide into sheet A has also recently been shown to dissipate electropositive surface potential present around the helix D region of P14-inserted ATIII (55).…”

“…Proteinase inhibitor function depends critically on mobility of the reactive loop, and in particular on its ability to stably insert into central ␤-sheet A during complex formation with target enzymes. Serpins in complex with their target proteinases are inferred to have their reactive loops partially inserted into their A-sheets as strand 4A (s4A) (3)(4)(5)(6). This partially inserted conformation is intermediate between those of native serpins, which have 5-stranded A-sheets (7,8), and latent and cleaved serpins which have fully 6-stranded A-sheets (9 -13).…”

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

“…Figure 2.12a shows that mAb # 2H5 bound at low titre to purified uPA:PAI-2 complex but not to either native (i.e stressed) PAI-2 nor uPA. It was expected that the formation of a complex between PAI-2 and the 14mer RCL peptide would mimic the interaction of PAI-2 with uPA by insertion of the RCL analogue into [3-sheet A of PAI-2, as previously observed with other serpins (Schulze et al 1990;Carrell et al 1991;Bjork et al 1992;Bjork 1993), hence inducing the relaxed conformation of the molecule. mAb # 2H5 binding to PAI-2/14mer RCL complexes was relatively high.…”

“…Using this approach, they estimated the conformational stabilit (AG[H 2 OJ) of inhibitory serpins in the S state to be 5 -15 kJ.mol" 1 , significantly lo than that of most globular proteins. Further evidence of large scale conformational changes accompanying the S->R transition is provided by immunological studies showing the expression of neoepitopes on a range of serpins following formation of stable serpin-protease complexes, Pl-Pl' cleavage, or insertion of synthetic RCL peptides (Schulze et al 1990;Bjork et al 1993;Dawes et al 1994;Eldering et al 1995;Debrock & Declerck 1995;Nordling & Bjork 1996).…”

“…As a member of the serpin family of proteins it is likely that PAI-2 undergoes the S->R conformational transition upon inhibition of its known target proteases (eg uPA, tPA), although this has never been experimentally verified. Detection of neoepitopes on complexes between antithrombin III and a synthetic RCL peptide (Bjork et al 1993) antithrombin-heparin complexes (Dawes et al 1994) and CI inhibitor-protease complexes (deAgostini et al 1988, Eldering et al 1995 suggests that immunological approaches can be utilised to discriminate between the conformational states associated with expression of serpin activity.…”