Latent Conformation Research Articles

Commercial plasma alpha1-antitrypsin (Prolastin) contains a conformationally inactive, latent component

Fractionated plasma alpha1-antitrypsin is widely-used as replacement therapy in patients with Z alpha1-antitrypsin deficiency-related emphysema. We have recently shown that purified antitrypsin may be induced to adopt an inactive latent conformation by heating at high temperatures in stabilizing concentrations of sodium citrate. Such a conformation was predicted to be present in commercial preparations of antitrypsin, as these require heating under similar conditions for viral inactivation. Native antitrypsin was purified from plasma, and commercial antitrypsin (Prolastin) was obtained from Bayer Corporation. Western blot analysis of transverse urea gradient (TUG) gels showed that commercial antitrypsin migrated as two bands: one with an unfolding profile of native antitrypsin and the second with a profile of latent antitrypsin. A latent fraction, comprising approximately 8% of the total antitrypsin, was separated from the native antitrypsin in Prolastin by anion exchange chromatography. The specific activity of this latent form against bovine alpha-chymotrypsin increased from 1 to 2% to 50% over 3 h after refolding from 6 M guanidine hydrochloride. These data show that commercial antitrypsin contains a latent component. The significance of this conformation in vivo is unknown, although Prolastin has shown few adverse side-effects in prolonged clinical usage.

Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two different mechanisms

Plasminogen activator inhibitor-1 (PAI-1), a unique member of the serpin superfamily, plays an important role in fibrinolysis and is an established risk factor for cardiovascular diseases. PAI-1 can occur in three interconvertible conformations: an active, a latent and a substrate form. To study conformational and functional relationships in PAI-1, a wide variety of monoclonal antibodies were evaluated for their influence on PAI-1 activity. Out of 77 monoclonal antibodies, directed against human PAI-1, six were selected for their strong inhibitory effect towards PAI-1 activity, i.e., 80 to 100% inhibition in the presence of a 1- to 16-fold molar excess of monoclonal antibody. Detailed analysis of the reaction products formed during the interaction between PAI-1 and its target proteinases tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA), in the presence of these monoclonal antibodies, revealed two distinct mechanisms of PAI-1 inactivation. Incubation of PAI-1 with one series of monoclonal antibodies resulted in the absence of any reaction indicative for direct interaction with the reactive-site loop or a facilitated conversion to the latent conformation. The loss of PAI-1 activity in the presence of the other group of monoclonal antibodies was associated with the concomitant formation of a 41 kDa cleavage product after interaction with the target proteinase. The latter observation demonstrates that binding of these antibodies induced a conformational change thereby converting the inhibitory, active conformation to the non-inhibitory substrate conformation. No conformational changes could be observed in latent PAI-1 under these conditions. Analysis of cross-reactivity revealed that some of these functionally important epitopes were conserved throughout PAI-1 obtained from various species including rabbit, mouse and/or pig, resulting in similar functional and conformational effects induced by these antibodies. Thus, we have demonstrated the occurrence of two distinct mechanisms by which the inhibitory activity of PAI-1 can be neutralized. This may have implications for the design of therapeutic or preventive strategies to interfere with PAI-1 activity. Cross-reactivity of these inhibitory antibodies with PAI-1 from various species may also allow their application in experimental animal models studying the in vivo role of PAI-I in various diseases (e.g. atherosclerosis, thrombosis, angiogenesis, …).

Kinetically controlled folding of the serpin plasminogen activator inhibitor 1.

The serpin plasminogen activator inhibitor 1 (PAI-1) folds into an active structure and then converts slowly to a more stable, but low-activity, "latent" conformation [Hekman, C. M., & Loskutoff, D. J. (1985) J. Biol. Chem. 260, 11581-11587]. Thus, the folding of PAI-1 is apparently under kinetic control. We have determined the urea denaturation and refolding transitions of both latent and active PAI-1 proteins by using intrinsic tryptophan fluorescence. While folding of active PAI-1 is reversible, the denaturation and refolding of latent PAI-1 are not. Instead, denatured latent PAI-1 refolds in lower concentrations of urea to give the active protein. Thus, the high-stability latent conformation is kinetically inaccessible over a range of urea concentrations. Complete denaturation of latent PAI-1 occurs at 5.5 M urea [delta G(H2O) approximately 21 kcal] whereas active PAI-1 denatures in only 3.8 M urea [delta G(H2O) approximately 12 kcal]. The fluorescence emission profile, as a function of urea of both the active and latent forms of the protein, reveals intermediates with partial structure. Circular dichroism measurements and limited protease digestion with Lys-C suggest that the intermediate in the denaturation of latent PAI-1 retains most of the secondary structure of the fully folded protein, whereas the intermediate in the denaturation of active PAI-1 exhibits significant loss of secondary structure. The Lys-C digestion patterns show that the active protein is more susceptible to proteolysis near sheet A than is the latent form. The studies suggest a model for the kinetically controlled folding pathway of PAI-1.

The Acid Stabilization of Plasminogen Activator Inhibitor-1 Depends on Protonation of a Single Group That Affects Loop Insertion into β-Sheet A

The serpin plasminogen activator inhibitor-1 (PAI-1) spontaneously adopts an inactive or latent conformation by inserting the N-terminal part of the reactive center loop as strand 4 into the major beta-sheet (sheet A). To examine factors that may regulate reactive loop insertion in PAI-1, we determined the inactivation rate of the inhibitor in the pH range 4.5-13. Below pH 9, inactivation led primarily to latent PAI-1, and one predominant effect of pH on the corresponding rate constant could be observed. Protonation of a group exhibiting a pKa of 7.6 (25 degrees C, ionic strength = 0.15 M) reduced the rate of formation of latent PAI-1 by a factor of 35, from 0.17 h-1 at pH 9 to about 0.005 h-1 below pH 6. The ionization with a pKa 7.6 was found to have no effect on the rate by which PAI-1 inhibits trypsin and is therefore unlikely to change the flexibility of the loop or the orientation of the reactive center. The peptides Ac-TEASSSTA and Ac-TVASSSTA (cf. P14-P7 in the reactive loop of PAI-1) formed stable complexes with PAI-1 and converted the inhibitor to a substrate for tissue type plasminogen activator. We found that peptide binding and formation of latent PAI-1 are mutually exclusive events, similarly affected by the pKa 7.6 ionization. This is direct evidence that external peptides can substitute for strand 4 in beta-sheet A of PAI-1 and that the pKa 7.6 ionization regulates insertion of complementary, internal or external, strands into this position. A model that accounts for the observed pH effects is presented, and the identity of the ionizing group is discussed based on the structure of latent PAI-1. The group is tentatively identified as His-143 in helix F, located on top of sheet A.

Mechanisms contributing to the conformational and functional flexibility of plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) is unique among the serine proteinase inhibitors (serpins) in that it can adopt at least three different conformations (active, substrate and latent). We report the X-ray structure of a cleaved substrate variant of human PAI-1, which has a new beta-strand s4A formed by insertion of the amino-terminal portion of the reactive-site loop into beta-sheet A subsequent to cleavage. This is in contrast to the previous suggestion that the non-inhibitory function of substrate-type serpins is mainly due to an inability of the reactive-site loop to adopt this conformation. Comparison with the structure of latent PAI-1 provides insights into the molecular determinants responsible for the transition of the stressed active conformation to the thermostable latent conformation.

Preparation and Characterization of Latent α1-Antitrypsin

Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A beta-pleated sheet and a mobile reactive center loop. The reactive center loop has been shown to adopt a range of conformations from the three turn alpha-helix of ovalbumin to the cleaved or latent inhibitor in which the reactive center loop is fully inserted into the A sheet of the molecule. While the cleaved state can be achieved in all inhibitory serpins only plasminogen activator inhibitor-1 and, more recently, antithrombin have been shown to adopt the latent conformation. We show here that the archetypal serpin, alpha 1-antitrypsin, can also be induced to adopt the latent conformation by heating at high temperatures in 0.7 M citrate for 12 h. The resulting species elutes at a lower sodium chloride concentration on an anion-exchange column and has a more cathodal electrophoretic mobility on non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing than native M antitrypsin. Latent antitrypsin is inactive as an inhibitor of bovine alpha-chymotrypsin, is stable to unfolding with 8 M urea, and is more resistant to heat-induced loop-sheet polymerization than native but less resistant than cleaved antitrypsin. The reactive center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sheet prevents the molecule accepting an exogenous reactive center loop peptide. The activity of latent antitrypsin may be increased from < 1% to approximately 35% by refolding from 6 M guanidinium chloride.

Serpins: implications of a mobile reactive centre.

The serpins are unique among the families of serine proteinase inhibitors in having a reactive centre that is situated on a mobile loop. The structures of three alternative conformations are now known, and it can be deduced that the active form involves the partial insertion of the loop into the A sheet of the molecule. The ability of the loop to move in and out of this sheet has been adapted by evolution to allow the modulation of inhibitory activity. Manipulation of the structure of the loop and of other functional domains in the serpin superfamily enables the production of serpins with tailor-made activities. The ability of the loop to lock in latent conformations or to take part in intermolecular polymerization has implications for the production and stabilization of recombinant serpins. This review has been adapted from Current Opinion in Structural Biology 1992, 2:438-446.