The length of the reactive center loop modulates the latency transition of plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three beta-sheets and several alpha-helices. Despite the similarity of its structure with those of other serpins, PAI-1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under physiological conditions. For the conformational conversion to occur, the reactive center loop (RCL) of PAI-1 must be mobilized and inserted into the major beta-sheet, A sheet. In an effort to understand how the structural conversion is regulated in this conformationally labile serpin, we modulated the length of the RCL of PAI-1. We show that releasing the constraint on the RCL by extension of the loop facilitates a conformational transition of PAI-1 to a stable state. Biochemical data strongly suggest that the stabilization of the transformed conformation is owing to the insertion of the RCL into A beta-sheet, as in the known latent form. In contrast, reducing the loop length drastically retards the conformational change. The results clearly show that the constraint on the RCL is a factor that regulates the conformational transition of PAI-1.     

Specific interactions of serpins in their native forms attenuate their conformational transitions

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein superfamily. Serpins are unique in that their native forms are not the most thermodynamically stable conformation; instead, a more stable, latent conformation exists. During the transition to the latent form, the first strand of beta-sheet C (s1C) in the serpin is peeled away from the beta-sheet, and the reactive center loop (RCL) is inserted into beta-sheet A, rendering the serpin inactive. To elucidate the contribution of specific interactions in the metastable native form to the latency transition, we examined the effect of mutations at the s1C of PAI-1, specifically in positions P4' through P10'. Several mutations strengthened the interactions between these residues and the core protein, and slowed the transition of the protein from the metastable native form to the latent form. In particular, anchoring of the strand to the protein's hydrophobic core at the beginning (P4' site) and center of the strand (P8' site) greatly retarded the latency transition. Mutations that weakened the interactions at the s1C region facilitated the conformational conversion of the protein to the latent form. PAI-1's overall structural stability was largely unchanged by the mutations, as evaluated by urea-induced equilibrium unfolding monitored via fluorescence emission. Therefore, the mutations likely exerted their effects by modulating the height of the energy barrier from the native to the latent form. Our results show that interactions found only in the metastable native form of serpins are important structural features that attenuate folding of the proteins into their latent forms.     

A peptide mimicking the C-terminal part of the reactive center loop induces the transition to the latent form of plasminogen activator inhibitor type-1

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of plasminogen activators (uPA and tPA) and thus plays a central role in fibrinolysis. The spontaneous insertion of its reactive centre loop (RCL) into β-sheet A is responsible for its irreversible conversion into the inactive latent form. In this study, we used two peptides mimicking residues P14-P9 and P8-P3 of the RCL so as to understand this dynamic process. We show that both peptides inhibit the formation of PAI-1/uPA and PAI-1/tPA complexes via two different mechanisms. Targeting the N-terminal part of the loop induces the cleavage of PAI-1 by the proteases uPA/tPA while targeting its C-terminal part greatly favors the irreversible formation of latent PAI-1.     

Accelerated conversion of human plasminogen activator inhibitor-1 to its latent form by antibody binding.

The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its beta-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA).PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1 labeled with the fluorescent probe N, N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9.NBD PAI-1 to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was protected from cleavage by elastase. The rate constants for MA-33B8 binding, measured by NBD fluorescence or inactivation, were similar (1.3-1.8 x 10(4) M-1 s-1), resulting in a 4000-fold faster inactivation at 4.2 microM antibody binding sites. The apparent antibody binding rate constant, at least 1000 times slower than one limited by diffusion, indicates that exposure of its epitope depends on an unfavorable equilibrium of PAI-1. Our observations are consistent with this idea and suggest that the equilibrium involves partial insertion of the RCL into sheet A: latent, RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted forms, bound much faster than active PAI-1 to MA-33B8, whereas two loop-extracted forms of PAI-1, modified to prevent loop insertion, did not bind or bound much more weakly to the antibody.     

Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation

The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A.The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered.The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta-sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A.The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin.     

Engineering of conformations of plasminogen activator inhibitor-1. A crucial role of beta-strand 5A residues in the transition of active form to latent and substrate forms.

The serpin (serine proteinase inhibitor) family is of general protein chemical interest because of its ability to undergo large conformational changes, in which the surface-exposed reactive centre loop (RCL) is inserted as strand 4 in the large central beta-sheet A. Loop insertion is an integral part of the inhibitory mechanism and also takes place at conversion of serpins to the latent state, occurring spontaneously only in plasminogen activator inhibitor-1 (PAI-1). We have investigated the importance of beta-strand 5A residues for the activity and latency transition of PAI-1. An approximately fourfold increase in the rate of latency transition resulted from His-substitution of Gln324 (position 334 in the alpha(1)-proteinase inhibitor template numbering), which interacts with the underlying alpha-helix B. The side chains of Gln321 and Lys325 (template residues 331 and 335, respectively) form hydrogen bonds to the peptide backbone of a loop connecting alpha-helix F and beta-strand 3A. While substitution with Ala of Glu321 had only minor effects on the properties of PAI-1, substitution with Ala of Lys325 led to stabilization of the inhibitory activity at incubation conditions leading to conversion of wild-type PAI-1 to a substrate form, and to an anomalous reaction towards a monoclonal antibody, which induced a delay in the latency transition of the mutant, but not wild-type PAI-1. We conclude that the anchoring of beta-strand 5A plays a crucial role in loop insertion. These findings provide new information about the mechanism of an important example of protein conformational changes.     

Mutational analysis of plasminogen activator inhibitor-1.

The serpin plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the plasminogen activating serine proteases tissue-type and urokinase-type plasminogen activator and, as such, an important regulator in turnover of extracellular matrix and in fibrinolysis. PAI-1 spontaneously loses its antiproteolytic activity by inserting its reactive centre loop (RCL) as strand 4 in beta-sheet A, thereby converting to the so-called latent state. We have investigated the importance of the amino acid sequence of alpha-helix F (hF) and the connecting loop to s3A (hF/s3A-loop) for the rate of latency transition. We grafted regions of the hF/s3A-loop from antithrombin III and alpha1-protease inhibitor onto PAI-1, creating eight variants, and found that one of these reversions towards the serpin consensus decreased the rate of latency transition. We prepared 28 PAI-1 variants with individual residues in hF and beta-sheet A replaced by an alanine. We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case. Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour, suggesting that insertion of the RCL is held back during latency transition as well as during complex formation with target proteases. The data presented identify new determinants of PAI-1 latency transition and provide general insight into the characteristic loop-sheet interactions in serpins.     

Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop

The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.     

The conversion of active to latent plasminogen activator inhibitor-1 is an energetically silent event

PAI-1 is a proteinase inhibitor, which plays a key role in the regulation of fibrinolysis. It belongs to the serpins, a family of proteins that behave either as proteinase inhibitors or proteinase substrates, both reactions involving limited proteolysis of the reactive center loop and insertion of part of this loop into beta-sheet A. Titration calorimetry shows that the inhibition of tissue-type plasminogen and pancreatic trypsin are exothermic reactions with DeltaH = -20.3, and -22.5 kcal.mol(-1), respectively. The Pseudomonas aeruginosa elastase-catalyzed reactive center loop cleavage and inactivation of the inhibitor is also exothermic (DeltaH = -38.9 kcal.mol(-1)). The bacterial elastase also hydrolyses peptide-bound PAI-1 in which acetyl-TVASSSTA, the octapeptide corresponding to the P(14)-P(7) sequence of the reactive center loop is inserted into beta-sheet A of the serpin with DeltaH = -4.0 kcal.mol(-1). In contrast, DeltaH = 0 for the spontaneous conversion of the metastable active PAI-1 molecule into its thermodynamically stable inactive (latent) conformer although this conversion also involves loop/sheet insertion. We conclude that the active to latent transition of PAI-1 is an entirely entropy-driven phenomenon.     

Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents.

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serpin family of serine proteinase inhibitors. Serpins inhibit their target proteinases by an ester bond being formed between the active site serine of the proteinase and the P1 residue of the reactive centre loop (RCL) of the serpin, followed by insertion of the RCL into beta-sheet A of the serpin. Concomitantly, there are conformational changes in the flexible joint region lateral to beta-sheet A. We have now, by site-directed mutagenesis, mapped the epitope for a monoclonal antibody, which protects the inhibitory activity of PAI-1 against inactivation by a variety of agents acting on beta-sheet A and the flexible joint region. Curiously, the epitope is localized in alpha-helix C and the loop connecting alpha-helix I and beta-strand 5A, on the side of PAI-1 opposite to beta-sheet A and distantly from the flexible joint region. By a combination of site-directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify a residue involved in conferring the antibody-induced conformational change from the epitope to the rest of the molecule. We have thus provided evidence for communication between secondary structural elements not previously known to interact in serpins.     

Structural differences between active forms of plasminogen activator inhibitor type 1 revealed by conformationally sensitive ligands

Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) in which the reactive center loop (RCL) spontaneously inserts into a central beta-sheet, beta-sheet A, resulting in inactive inhibitor. Available x-ray crystallographic studies of PAI-1 in an active conformation relied on the use of stabilizing mutations. Recently it has become evident that these structural models do not adequately explain the behavior of wild-type PAI-1 (wtPAI-1) in solution. To probe the structure of native wtPAI-1, we used three conformationally sensitive ligands: the physiologic cofactor, vitronectin; a monoclonal antibody, 33B8, that binds preferentially to RCL-inserted forms of PAI-1; and RCL-mimicking peptides that insert into beta-sheet A. From patterns of interaction with wtPAI-1 and the stable mutant, 14-1B, we propose a model of the native conformation of wtPAI-1 in which the bottom of the central sheet is closed, whereas the top of the beta-sheet A is open to allow partial insertion of the RCL. Because the incorporation of RCL-mimicking peptides into wtPAI-1 is accelerated by vitronectin, we further propose that vitronectin alters the conformation of the RCL to allow increased accessibility to beta-sheet A, yielding a structural hypothesis that is contradictory to the current structural model of PAI-1 in solution and its interaction with vitronectin.     

Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.

The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.     

Extending the capabilities of targeted molecular dynamics: simulation of a large conformational transition in plasminogen activator inhibitor 1

Plasminogen activator inhibitor type 1 (PAI-1) is an inhibitor of plasminogen activators such as tissue-type plasminogen activator or urokinase-type plasminogen activator. For this molecule, different conformations are known. The inhibiting form that interacts with the proteinases is called the active form. The noninhibitory, noncleavable form is called the latent form. X-ray and modeling studies have revealed a large change in position of the reactive center loop (RCL), responsible for the interaction with the proteinases, that is inserted into a beta-sheet (s4A) in the latent form. The mechanism underlying this spontaneous conformational change (half-life = 2 h at 37 degrees C) is not known in detail. This investigation attempts to predict a transition path from the active to the latent structure at the atomic level, by using simulation techniques. Together with targeted molecular dynamics (TMD), a plausible assumption on a rigid body movement of the RCL was applied to define an initial guess for an intermediate. Different pathways were simulated, from the active to the intermediate, from the intermediate to the latent structure and vice versa under different conditions. Equilibrium simulations at different steps of the path also were performed. The results show that a continuous pathway from the active to the latent structure can be modeled. This study also shows that this approach may be applied in general to model large conformational changes in any kind of protein for which the initial and final three-dimensional structure is known.     

Polymerization of plasminogen activator inhibitor-1

The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into beta-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and beta-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-A-sheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving beta-strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet.     

Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.     

Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A

The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the serpin structure involving rapid insertion of the RCL into the center of an existing beta-sheet, serpin beta-sheet A. The present study demonstrates that partitioning between inhibitor and substrate modes of reaction can be altered by varying either the rates of RCL insertion or deacylation using a library of serpin RCL mutants substituted in the critical P(14) hinge residue and three different proteinases. We further correlate the changes in partitioning with the actual rates of RCL insertion for several of the variants upon reaction with the different proteinases as determined by fluorescence spectroscopy of specific RCL-labeled inhibitor mutants. These data demonstrate that the serpin mechanism follows a branched pathway, and that the formation of a stable inhibited complex is dependent upon both the rate of the RCL conformational change and the rate of enzyme deacylation.     

Structural factors affecting the choice between latency transition and polymerization in inhibitory serpins

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) protein family, is unique among the serpins in its conformational lability. This lability allows spontaneous conversion of the active form to a more stable, latent conformation under physiological conditions. In other serpins, polymerization, rather than latency transition, is induced under pathological conditions or upon heat treatment. To identify specific factors promoting latency conversion in PAI-1, we mutated PAI-1 at various positions and compared the effects with those of equivalent mutations in alpha(1)-antitrypsin, the archetypal serpin. Mutations that improved interactions with the turn between helix F and the third strand of beta-sheet A (thFs3A) or the fifth strand of beta-sheet A (s5A), which are near the site of latency transition-associated insertion of the reactive center loop, retarded latency conversion but did not greatly increase structural stability. Mutations that decreased interactions with s2C facilitated conformational conversion, possibly by releasing the reactive center loop from beta-sheet C. Mutations of Thr93 that filled a hydrophobic surface pocket on s2A dramatically increased structural stability but had a negligible effect on the conformational transition. Our results suggest that the structural features controlling latency transition in PAI-1 are highly localized, whereas the conformational strain of the native forms of other inhibitory serpins is distributed throughout the molecule and induces polymerization.     

The contribution of the exosite residues of plasminogen activator inhibitor-1 to proteinase inhibition

The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and beta-trypsin by PAI-1. Alanine substitutions at the distal P4' (Glu-350) and P5' (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k(a)) and overall inhibition (k(app)) with tPA by 13.4- and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k(lim)) increased by 3.3-fold. The effects of double mutations on k(a), k(lim), and k(app) were small with uPA and nonexistent with beta-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into beta-sheet A. Moreover, mutational analysis indicates that the P5' residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.     

Mapping of a conformational epitope on plasminogen activator inhibitor-1 by random mutagenesis. Implications for serpin function

The mechanism for the conversion of plasminogen activator inhibitor-1 (PAI-1) from the active to the latent conformation is not well understood. Recently, a monoclonal antibody, 33B8, was described that rapidly converts PAI-1 to the latent conformation (Verhamme, I., Kvassman, J. O., Day, D., Debrock, S., Vleugels, N., Declerck, P. J., and Shore, J. D. (1999) J. Biol. Chem. 274, 17511-17517). In an attempt to understand this interaction, and more broadly to understand the mechanism of the natural transition of PAI-1 to the latent conformation, we have used random mutagenesis to identify the 33B8 epitope in PAI-1. This site involves at least 8 amino acids scattered over more than two-thirds of the linear sequence that form a compact epitope on the PAI-1 three-dimensional structure. Surface plasmon resonance studies indicate a high affinity interaction between latent PAI-1 and 33B8 that is approximately 100-fold higher than comparable binding to active PAI-1. Structural modeling results together with surface plasmon resonance analysis of parental and site-directed PAI-1 mutants with disrupted 33B8 binding suggest the existence of a specific PAI-1 intermediate structure that is stabilized by 33B8 binding. These analyses strongly suggest that this intermediate form of PAI-1 has a partial insertion of the reactive center loop into beta-sheet A, and together, these data have significant implications for the general serpin mechanism of proteinase inhibition.     

Latency and substrate binding globally reduce solvent accessibility of plasminogen activator inhibitor type 1 (PAI-1). An adaptation of PAI-1 conformer crystal structures by hydrogen-deuterium exchange

Plasminogen activator inhibitor type 1 (PAI-1) plays key regulatory roles in fibrinolysis, cell migration, and tissue remodeling. A regulatory protein without known catalytic activity, PAI-1 modulates plasminogen activators through protein-protein interactions. Although global conformational alterations that occur in PAI-1 determine its regulatory activity, comprehensive assessments of concurrent dynamic, structural, and functional alterations of this critical regulatory protein have not yet been clearly defined. X-ray crystallographic studies have described four distinct PAI-1 conformational states: active, latent, reactive center loop peptide-annealed (RCL-PA), and cleaved mutant. In this study, backbone amide hydrogen-deuterium exchange detected by mass spectrometry was used to characterize dynamic and structural alterations of human PAI-1 (hPAI-1) in relation to its function. hPAI-1 conformers were defined by surface mapping the solvent-accessible sites for strategic secondary structural components of the protein. We observed a global protection from solvent for a majority of peptides in the latent conformer relative to the active conformer. Significant differences were observed in the RCL, helix A, helix D, and sheet 1C, and these regions were markedly more dynamic or solvent-exposed in the active conformation. The RCL-PA form adopts an intermediate conformational state between the active and the latent conformers. Our results demonstrate that the most dynamic regions of PAI-1 (the RCL, helices D and A, and sheet 5A) are flexible in the transition toward latency. They also show that the dynamic surface structures of the active, latent, and peptide-annealed conformers of PAI-1 are underestimated by theoretical solvent accessibility calculations derived from crystallographic data.