Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs. A study by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy.

Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cooperative unfolding of a metastable serpin to a molten globule suggests a link between functional and folding energy landscapes

Alpha-1 antitrypsin (alpha(1)-AT) is a member of the serpin class of protease inhibitors, and folds to a metastable state rather than its thermodynamically most stable native state. Upon cleavage by a target protease, alpha(1)-AT undergoes a dramatic conformational change to a stable form, translocating the bound protease more than 70 A to form an inhibitory protease-serpin complex. Numerous mutagenesis studies on serpins have demonstrated the trade-off between the stability of the metastable state on the one hand and the inhibitory efficiency on the other. Studies of the equilibrium unfolding of serpins provide insight into this connection between structural plasticity and metastability. We studied equilibrium unfolding of wild-type alpha(1)-AT using hydrogen-deuterium/exchange mass spectrometry to characterize the structure and the stability of an equilibrium intermediate that was observed in low concentrations of denaturant in earlier studies. Our results show that the intermediate observed at low concentrations of denaturant has no protection from hydrogen-deuterium exchange, indicating a lack of stable structure. Further, differential scanning calorimetry of alpha(1)-AT at low concentrations of denaturant shows no heat capacity peak during thermal denaturation, indicating that the transition from the intermediate to the unfolded state is not a cooperative first-order-like phase transition.. Our results show that the unfolding of alpha(1)-AT involves a cooperative transition to a molten globule form, followed by a non-cooperative transition to a random-coil form as more guanidine is added. Thus, the entire alpha(1)-AT molecule consists of one cooperative structural unit rather than multiple structural domains with different stabilities. Furthermore, our results together with previous mutagenesis studies suggest a possible link between an equilibrium molten globule and a functional intermediate that may be populated during the protease inhibition.     

The structural basis of serpin polymerization studied by hydrogen/deuterium exchange and mass spectrometry

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type alpha(1)-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the alpha(1)-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of beta-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into beta-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of beta-sheet C. On the basis of our results, we propose a mechanism for polymerization in which beta-strand 1C is displaced from the rest of beta-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of beta-sheet A, and allows for subsequent polymerization.     

Comparative fourier transform infrared and circular dichroism spectroscopic analysis of alpha1-proteinase inhibitor and ovalbumin in aqueous solution

Alpha1-proteinase inhibitor (alpha1Pi) and ovalbumin are both members of the serpin superfamily. They share about a 30% sequence identity and exhibit great similarity in their three-dimensional structures. However, no apparent functional relationship has been found between the two proteins. Unlike alpha1Pi, ovalbumin shows no inhibitory effect to serine proteases. To see whether or not a conformational factor(s) may contribute to the functional difference, we carried out comparative analysis of the two proteins' secondary structure, thermal stability, and H-D exchange using FT-IR and CD spectroscopy. FT-IR analysis reveals significant differences in the amide I spectral patterns of the two proteins. Upon thermal denaturation, both proteins exhibit a strong low-wavenumber beta-sheet band at 1624 cm(-1) and a weak high-wavenumber beta-sheet band at 1694 cm(-1), indicative of intermolecular aggregate formation. However, the midpoint of the thermal-induced transition of alpha1Pi (approximately 55 degrees C) is 18 degrees C lower than that of ovalbumin (approximately 73 degrees C). The thermal stability analysis provides new insight into the structural changes associated with denaturation. The result of H-D exchange explains some puzzling spectral differences between the two proteins in D2O reported previously.     

Structural comparisons of the native and reactive-centre-cleaved forms of alpha 1-antitrypsin by neutron- and X-ray-scattering in solution.

alpha 1-Antitrypsin is the best-characterized member of the serpin (serine-proteinase inhibitor) superfamily. Its solution structure was studied by high-flux neutron-scattering and synchrotron X-ray-scattering. Neutron data show that its absorption coefficient A1% 280,1cm is 5.4. The neutron radius of gyration RG at infinite contrast for native alpha 1-antitrypsin is 2.61 nm, characteristic of a moderately elongated structure, and its cross-sectional RG is 1.34 nm. The internal inhomogeneity of scattering densities within alpha 1-antitrypsin is high at 29 x 10(-5). The X-ray RG is 2.91 nm, in good agreement with the neutron RG of 2.82 nm in 1H2O. This RG is unchanged in reactive-centre-cleaved alpha 1-antitrypsin. These parameters are also unchanged at pH 8 in sodium/potassium phosphate buffers up to 0.6 M. The neutron and X-ray curves for native alpha 1-antitrypsin were compared with Debye simulation based on the crystal structure of reactive-centre-cleaved (papain) alpha 1-antitrypsin. After allowance for residues not visible in the crystallographic electron-density map, and rejoining the proteolysed site between Met-358 and Ser-359 by means of a relatively minor conformational re-arrangement, good agreement to a structural resolution of 4 nm is obtained with the neutron data in two contrasts and with the X-ray data. The structures of the native and cleaved forms of alpha 1-antitrypsin are thus similar within the resolution of solution scattering. This places an upper limit on the magnitude of the presumed conformational changes that occur in alpha 1-antitrypsin on reactive-centre cleavage, as indicated in earlier spectroscopic investigations of the Met-358-Ser-359 peptide-bond cleavage. Methods for scattering-curve simulations from crystal structures are critically assessed. The RG data lead to dimensions of 7.8 nm x 4.9 nm x 2.2 nm for native alpha 1-antitrypsin. The high internal inhomogeneity and the asymmetric shorter semi-axes of 4.9 nm and 2.2 nm suggest that the three oligosaccharide chains of alpha 1-antitrypsin are essentially freely extended into solvent in physiological conditions. This conclusion is also supported by the Debye simulations, and by modelling based on hydrodynamic parameters.     

Fluorescence correlation spectroscopic study of serpin depolymerization by computationally designed peptides

Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within beta-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the beta-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within beta-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of alpha(1)-antitrypsin (alpha(1)-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found.     

Role of the connectivity of secondary structure segments in the folding of alpha(1)-antitrypsin

The native form of serpins (serine protease inhibitors) is metastable, which is critical to their biological functions. Spontaneous conversion from the native form of serpins into a more stable conformation, called the "latent" form, is restricted. To examine whether the connectivity of strand 1 of beta-sheet C to the hydrophobic core is critical to the serpin's preferential folding to the metastable native conformation, we designed a circularly-permuted mutant of alpha(1)-antitrypsin, the prototype serpin, in which strand 1C is disconnected from the hydrophobic core. Conformation of the circular permutant was similar to that of the latent form, as revealed by equilibrium unfolding, limited proteolysis, and spectroscopic properties. Our results support the notion that rapid folding of the hydrophobic core with concomitant incorporation of strand 1C into beta-sheet C traps the serpin molecule into its native metastable conformation.     

alpha1-Proteinase inhibitor forms initial non-covalent and final covalent complexes with elastase analogously to other serpin-proteinase pairs, suggesting a common mechanism of inhibition

Despite several concordant structural studies on the initial non-covalent complex that serpins form with target proteinases, a recent study on the non-covalent complex between the serpin alpha(1)-proteinase inhibitor (alpha(1)PI) and anhydroelastase concluded that translocation of the proteinase precedes cleavage of the reactive center loop and formation of the acyl ester. Because this conclusion is diametrically opposite to those of the other structural studies on serpin-proteinase pairs, we proceeded to examine this specific serpin-proteinase complex by the same successful NMR approach used previously on the alpha(1)PI-Pittsburgh-S195A trypsin pair. Both non-covalent complex with anhydroelastase and covalent complex with active elastase were made with (15)N-alanine-labeled wild-type alpha(1)PI. The heteronuclear single quantum correlation spectroscopy (HSQC) NMR spectrum of the non-covalent complex showed that the entire reactive center loop remained exposed, and the serpin body maintained a conformation indistinguishable from that of native alpha(1)PI, indicating no movement of the proteinase and no insertion of the reactive center loop into beta-sheet A. In contrast, the HSQC NMR spectrum of the covalent complex showed that the reactive center loop had fully inserted into beta-sheet A, indicating that translocation of the proteinase had occurred. These results agree with previous NMR, fluorescence resonance energy transfer, and x-ray crystallographic studies and suggest that a common mechanism is employed in formation of serpin-proteinase complexes. We found that preparations of anhydroelastase that are not appropriately purified contain material that can regenerate active elastase over time. It is likely that the material used by Mellet and Bieth contained such active elastase, resulting in mistaken attribution of the behavior of covalent complex to that of the non-covalent complex.     

Interactions causing the kinetic trap in serpin protein folding

Conformational transition is fundamental to the mechanism of functional regulation in proteins, and serpins (serine protease inhibitors) can provide insight into this process. Serpins are metastable in their native forms, and they ordinarily undergo conformational transition to a stable state only when they form a tight complex with target proteases. The metastable native form is thus considered to be a kinetically trapped folding intermediate. We sought to understand the nature of the serpin kinetic trap as a step toward discovering how conformational transition is regulated. We found that mutations of the B/C beta-barrel of native alpha(1)-antitrypsin, a prototypical serpin, allowed conversion of the molecule into a more stable state. A 2.2 A resolution crystal structure of the stable form (PDB code, ) showed that the reactive site loop is inserted into an A beta-sheet, as in the latent plasminogen activator inhibitor-1. Mutational analyses suggest strongly that interactions not found in the final stable form cause the kinetic trap in serpin protein folding.     

Probing the equilibrium denaturation of the serpin alpha(1)-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state.

The native conformation of proteins in the serpin superfamily is metastable. In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of alpha(1)-antitrypsin. alpha(1)-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B beta-sheets, respectively. Site-directed mutagenesis was used to construct two single-tryptophan variants. Both variants were fully active and had similar secondary structure and stabilities to alpha(1)-antitrypsin. The denaturation of alpha(1)-antitrypsin and its variants was extremely similar when followed by far-UV CD, indicating the presence of a single intermediate. Fluorescence analysis of the unfolding behavior of each single tryptophan variant indicated that the sole tryptophan residue reported the structural changes within its immediate environment. These data suggest that the A beta-sheet is expanded in the intermediate state whilst no structural change around the B beta-sheet has occurred. In the urea-induced unfolded state, Trp238 does not become fully solvated, suggesting the persistence of structure around this residue. The implications of these data on the folding, misfolding and function of the serpin superfamily are discussed.     

Photo-induced unfolding and inactivation of bovine carbonic anhydrase in the presence of a photoresponsive surfactant

Photoreversible changes in the conformation and enzymatic activity of bovine carbonic anhydrase have been investigated as a function of photoresponsive surfactant concentration and light conditions. The light-responsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to photoreversibly control enzyme–surfactant interactions. Small-angle neutron scattering and dynamic light scattering measurements, along with fluorescence spectroscopy, indicate that carbonic anhydrase unfolds upon addition of the surfactant under visible light, while only a small degree of unfolding is observed under UV light. Therefore, the enzyme is completely inactivated in the presence of the trans surfactant, while 40% of the native activity is preserved under UV light, providing a photoreversible “on/off switch” of enzyme activity. Small-angle neutron scattering data provide details of the in vitro conformational changes of the enzyme in response to the photosurfactant and light, with the enzyme found to aggregate as a result of photosurfactant-induced unfolding. Fourier transform infrared (FT-IR) spectroscopy further provides information on the secondary structure changes of the protein in the presence of photosurfactant.     

Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.     

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.     

Cleaved serpin refolds into the relaxed state via a stressed conformer

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1' bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1'-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly(1)-Ala(352)) and light (Ser(353)-Pro(385)) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T(m) = 68.4 degrees C, whereas cl-R339T refolded into the relaxed form with T(m) = 85.5 degrees C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1'-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure.     

Inhibition of activated protein C by recombinant alpha 1-antitrypsin variants with substitution of arginine or leucine for methionine358

alpha 1-Antitrypsin (alpha 1-AT) was recently identified as a major physiologic plasma inhibitor of activated protein C. The reaction with activated protein C of recombinant alpha 1-AT containing amino acid substitutions at the reactive center was studied. The substitution of Arg358 for Met, as observed in a patient with a severe bleeding disorder with the mutant alpha 1-AT Pittsburgh, increased the association rate constant for activated protein C from 1.1 x 10(1) to 4.9 x 10(4) M-1 s-1. The association rate constant of activated protein C with protein C inhibitor, a native plasma serpin that contains Arg354 at the reactive site, is 6 x 10(3) M-1 s-1 in the absence of heparin. Plasma containing 4 microM [Arg358]alpha 1-AT inhibited activated protein C activity by greater than 95% in 15 s, and the inhibited activated protein C was shown by immunoblotting to exist as activated protein C-inhibitor complexes. In controls 50% loss of activated protein C activity in normal plasma occurred in 19 min. Double-substituted [Pro357,Met358]alpha 1-AT----[Ala357,Arg358]alpha 1-AT had similar reactivity toward activated protein C as the single-substituted [Arg358]alpha 1-AT. Thus, replacement of the reactive center Met358 of alpha 1-AT by Arg358, analogous to Arg354 of protein C inhibitor, results in an activated protein C inhibitor that is more potent than either of the native inhibitors. Comparison of the association rate constant of the [Arg358]alpha 1-AT for activated protein C to that for thrombin (4 x 10(4) versus 3 x 10(5) M-1 s-1) suggests that thrombin would be more effectively inhibited than activated protein C, thereby giving an explanation for bleeding rather than thrombosis in the alpha 1-AT Pittsburgh patient.     

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 2.6 A structure of a serpin polymer and implications for conformational disease.

The function of the serpins as proteinase inhibitors depends on their ability to insert the cleaved reactive centre loop as the fourth strand in the main A beta-sheet of the molecule upon proteolytic attack at the reactive centre, P1-P1'. This mechanism is vulnerable to mutations which result in inappropriate intra- or intermolecular loop insertion in the absence of cleavage. Intermolecular loop insertion is known as serpin polymerisation and results in a variety of diseases, most notably liver cirrhosis resulting from mutations of the prototypical serpin alpha1-antitrypsin. We present here the 2.6 A structure of a polymer of alpha1-antitrypsin cleaved six residues N-terminal to the reactive centre, P7-P6 (Phe352-Leu353). After self insertion of P14 to P7, intermolecular linkage is affected by insertion of the P6-P3 residues of one molecule into the partially occupied beta-sheet A of another. This results in an infinite, linear polymer which propagates in the crystal along a 2-fold screw axis. These findings provide a framework for understanding the uncleaved alpha1-antitrypsin polymer and fibrillar and amyloid deposition of proteins seen in other conformational diseases, with the ordered array of polymers in the crystal resulting from slow accretion of the cleaved serpin over the period of a year.     

Topography of a 2.0 A structure of alpha1-antitrypsin reveals targets for rational drug design to prevent conformational disease

Members of the serpin family of serine proteinase inhibitors play important roles in the inflammatory, coagulation, fibrinolytic, and complement cascades. An inherent part of their function is the ability to undergo a structural rearrangement, the stressed (S) to relaxed (R) transition, in which an extra strand is inserted into the central A beta-sheet. In order for this transition to take place, the A sheet has to be unusually flexible. Malfunctions in this flexibility can lead to aberrant protein linkage, serpin inactivation, and diseases as diverse as cirrhosis, thrombosis, angioedema, emphysema, and dementia. The development of agents that control this conformational rearrangement requires a high resolution structure of an active serpin. We present here the topology of the archetypal serpin alpha1-antitrypsin to 2 A resolution. This structure allows us to define five cavities that are potential targets for rational drug design to develop agents that will prevent conformational transitions and ameliorate the associated disease.     

The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.