S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The structure of active serpin 1K from Manduca sexta.

BACKGROUND: The reactive center loops (RCL) of serpins undergo large conformational changes triggered by the interaction with their target protease. Available crystallographic data suggest that the serpin RCL is polymorphic, but the relevance of the observed conformations to the competent active structure and the conformational changes that occur on binding target protease has remained obscure. New high-resolution data on an active serpin, serpin 1K from the moth hornworm Manduca sexta, provide insights into how active serpins are stabilized and how conformational changes are induced by protease binding. RESULTS: The 2.1 A structure shows that the RCL of serpin 1K, like that of active alpha1-antitrypsin, is canonical, complimentary and ready to bind to the target protease between P3 and P3 (where P refers to standard protease nomenclature),. In the hinge region (P17-P13), however, the RCL of serpin 1K, like ovalbumin and alpha1-antichymotrypsin, forms tight interactions that stabilize the five-stranded closed form of betasheet A. These interactions are not present in, and are not compatible with, the observed structure of active alpha1-antitrypsin. CONCLUSIONS: Serpin 1K may represent the best resting conformation for serpins - canonical near P1, but stabilized in the closed conformation of betasheet A. By comparison with other active serpins, especially alpha1-antitrypsin, a model is proposed in which interaction with the target protease near P1 leads to conformational changes in betasheet A of the serpin.     

Expression and characterization of recombinant Manduca sexta serpin- 1B and site-directed mutants that change its inhibitory selectivity

Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily. During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin. The amino acid residue on the amino-terminal side of this scissile bond, the P₁ residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases. M. sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P₁ residue, was previously named alaserpin. This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F). The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli. Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin. All three serpins inhibited human cathepsin G. This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins. Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited. The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343. These results demonstrate that the P₁ alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position.     

Ovalbumin and angiotensinogen lack serpin S-R conformational change.

Cleavage of ovalbumin and angiotensinogen at sites homologous to the reactive centre loop of alpha 1-antitrypsin is not accompanied by the increase in heat-stability associated with the transition from the native stressed (S) structure to a cleaved relaxed (R) form that is typical of other serpins. Failure to undergo the S-R change in ovalbumin is not due to phosphorylation of Ser-344 near the sites of cleavage on the loop. The suggested explanation is the unique presence of bulky side chains at the P10-P12 site in ovalbumin and angiotensinogen.     

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.     

Loop-inserted and thermostabilized structure of P1-P1' cleaved ovalbumin mutant R339T.

Ovalbumin is a member of a superfamily of serine proteinase inhibitors, known as the serpins. It is, however, non-inhibitory towards serine proteinases, and lacks the loop insertion mechanism common to the serpins due to unknown structural factors. Mutant ovalbumin, R339T, in which the P14 hinge residue is replaced, was produced and analyzed for its thermostability and three-dimensional structure. Differential scanning calorimetry revealed that the mutant ovalbumin, but not the wild-type protein, undergoes a marked thermostabilization (DeltaT(m)=15.8 degrees C) following the P1-P1' cleavage. Furthermore, the crystal structure, solved at 2.3 A resolution, clearly proved that the P1-P1' cleaved form assumes the fully loop-inserted conformation as seen in serpin that possess inhibitory activity. We therefore conclude that ovalbumin acquires the structural transition mechanism into the loop-inserted, thermostabilized form by the single hinge mutation. The mutant protein does not, however, possess inhibitory activity. The solved structure displays the occurrence of specific interactions that may prevent the smooth motion, relative to sheet A, of helices E and F and of the loop that follows helix F. These observations provide crucial insights into the question why R339T is still non-inhibitory.     

Conformational changes in serpins: I. The native and cleaved conformations of alpha(1)-antitrypsin

The serpins (SERine Proteinase INhibitors) are a family of proteins with important physiological roles, including but not limited to the inhibition of chymotrypsin-like serine proteinases. The inhibitory mechan- ism involves a large conformational change known as the S-->R (stressed-->relaxed) transition. The largest structural differences occur in a region around the scissile bond called the reactive centre loop: In the native (S) state, the reactive centre is exposed, and is free to interact with proteinases. In inhibitory serpins, in the cleaved (R) state the reactive centre loop forms an additional strand within the beta-sheet. The latent state is an uncleaved state in which the intact reactive centre loop is integrated into the A sheet as in the cleaved form, to give an alternative R state.The serpin structures illustrate detailed control of conformation within a single protein. Serpins are also an unusual family of proteins in which homologues have native states with different folding topologies. Determination of the structures of inhibitory serpins in multiple conformational states permits a detailed analysis of the mechanism of the S-->R transition, and of the way in which a single sequence can form two stabilised states of different topology.Here we compare the conformations of alpha(1)-antitrypsin in native and cleaved states. Many protein conformational changes involve relative motions of large rigid subunits. We determine the rigid subunits of alpha(1)-antitrypsin and analyse the changes in their relative position and orientation. Knowing that the conformational change is initiated by cleavage at the reactive centre, we describe a mechanism of the S-->R transition as a logical sequence of mechanical effects, even though the transition likely proceeds in a concerted manner.     

A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.     

Conformational changes in serpins: I. The native and cleaved conformations of alpha(1)-antitrypsin

The serpins (SERine Proteinase INhibitors) are a family of proteins with important physiological roles, including but not limited to the inhibition of chymotrypsin-like serine proteinases. The inhibitory mechan- ism involves a large conformational change known as the S-->R (stressed-->relaxed) transition. The largest structural differences occur in a region around the scissile bond called the reactive centre loop: In the native (S) state, the reactive centre is exposed, and is free to interact with proteinases. In inhibitory serpins, in the cleaved (R) state the reactive centre loop forms an additional strand within the beta-sheet. The latent state is an uncleaved state in which the intact reactive centre loop is integrated into the A sheet as in the cleaved form, to give an alternative R state.The serpin structures illustrate detailed control of conformation within a single protein. Serpins are also an unusual family of proteins in which homologues have native states with different folding topologies. Determination of the structures of inhibitory serpins in multiple conformational states permits a detailed analysis of the mechanism of the S-->R transition, and of the way in which a single sequence can form two stabilised states of different topology.Here we compare the conformations of alpha(1)-antitrypsin in native and cleaved states. Many protein conformational changes involve relative motions of large rigid subunits. We determine the rigid subunits of alpha(1)-antitrypsin and analyse the changes in their relative position and orientation. Knowing that the conformational change is initiated by cleavage at the reactive centre, we describe a mechanism of the S-->R transition as a logical sequence of mechanical effects, even though the transition likely proceeds in a concerted manner.     

A 1H NMR probe for mobility in the reactive center loops of serpins: spin-echo studies of native and modified forms of ovalbumin and alpha 1-proteinase inhibitor

It has recently been proposed that the expression of inhibitory activity in serine protease inhibitors (serpins) is a function of the mobility of the extended alpha-helical reactive center loop [Stein, P.E., Leslie, A.G.W., Finch, J.T., Turnell, W.G., McLaughlin, P.J., & Carrell, R.W. (1990) Nature 347, 99-102]. We have employed solution 1H NMR methods, including the Carr-Purcell-Meiboom-Gill (CPMG) and Hahn spin-echo pulse sequences, to try to identify such regions by virtue of their anticipated longer T2 relaxation times in two of the best characterized members of the serpin superfamily, ovalbumin and alpha 1-proteinase inhibitor. The CPMG spectra of native ovalbumin reveal the presence of long-lived resonances from the methyl protons of alanine residues and the CH3 protons of leucine or valine residues as well as the acetyl and ring methine protons of the carbohydrate moieties. Following reaction of ovalbumin with subtilisin Carlsberg to generate plakalbumin [where excision from within the reactive center loop homologue of a hexa- or heptapeptide, with sequence (E)-A-G-V-D-A-A, occurs], its CPMG spectrum retained almost all of the originally present long-lived resonances. Concurrent with the retention of these mobile resonances in plakalbumin is the appearance of two additional resonances consistent with the formation of new C and N termini. On the basis of the proposed mobility of the reactive center loop, it had been expected that removal of the alanine-rich hexapeptide would result in loss of some or all of the long-lived alanine methyl resonances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of a noncovalent serpin-proteinase complex involves no conformational change in the serpin. Use of 1H-15N HSQC NMR as a sensitive nonperturbing monitor of conformation

A structural understanding of the nature and scope of serpin inhibition mechanisms has been limited by the inability so far to crystallize any serpin-proteinase complex. We describe here the application of [(1)H-(15)N]-HSQC NMR on uniformly and residue-selectively (15)N-labeled serpin alpha(1)-proteinase inhibitor (Pittsburgh variant with stabilizing mutations) to provide a nonperturbing and exquisitely sensitive means of probing the conformation of the serpin alone and in a noncovalent complex with inactive, serine 195-modified, bovine trypsin. The latter should be a good model both for the few examples of reversible serpin-proteinase complexes and for the initial Michaelis-like complex formed en route to irreversible covalent inhibition. Cleavage of the reactive center loop, with subsequent insertion into beta-sheet A, caused dramatic perturbation of most of the NMR cross-peaks. This was true for both the uniformly labeled and alanine-specifically labeled samples. The spectra of uniformly or leucine- or alanine-specifically labeled alpha(1)-proteinase inhibitor in noncovalent complex with unlabeled inactive trypsin gave almost no detectable chemical shift changes of cross-peaks, but some general increase in line width. Residue-specific assignments of the four alanines in the reactive center loop, at P12, P11, P9, and P4, allowed specific examination of the behavior of the reactive center loop. All four alanines showed higher mobility than the body of the serpin, consistent with a flexible reactive center loop, which remained flexible even in the noncovalent complex with proteinase. The three alanines near the hinge point for insertion showed almost no chemical shift perturbation upon noncovalent complex formation, while the alanine at P4 was perturbed, presumably by interaction with the active site of bound trypsin. Reporters from both the body of the serpin and the reactive center loop therefore indicate that noncovalent complex formation involves no conformational change in the body of the serpin and only minor perturbation of the reactive center loop in the region which contacts proteinase. Thus, despite the large size of serpin and serpin-proteinase complex, 45 and 69 kDa respectively, NMR provides a very sensitive means of probing serpin conformation and mobility, which should be applicable both to noncovalent and to covalent complexes with a range of different proteinases, and probably to other serpins.     

Probing the local conformational change of alpha1-antitrypsin

The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of beta-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in alpha(1)-antitrypsin, which would lock the displacement of thFs3A from beta-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop-sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop-sheet polymerization significantly. This successful protein engineering of alpha(1)-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into beta-sheet A while a substantial portion of the interactions between hF and s3A is kept intact.     

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.     

Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substrates.

The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747-2754]. Selected substrates were then transplanted into the reactive site loop of alpha1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific alpha1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.     

Cavities of α1-antitrypsin that play structural and functional roles

The native form of inhibitory serine protease inhibitors (serpins) is strained, which is critical for their inhibitory activity. Previous studies on stabilizing mutations of alpha(1)-antitrypsin, a prototype of serpins, indicated that cavities provide a structural basis for the native strain of the molecule. We have systematically mapped the cavities of alpha(1)-antitrypsin that play such structural and functional roles by designing cavity-filling mutations at residues that line the walls of the cavities. Results show that energetically unfavorable cavities are distributed throughout the alpha(1)-antitrypsin molecule, and the cavity-filling mutations stabilized the native conformation at 8 out of 10 target sites. The stabilization effect of the individual cavity-filling mutations of alpha(1)-antitrypsin varied (0.2-1.9 kcal/mol for each additional methylene group) and appeared to depend largely on the structural flexibility of the cavity environment. Cavity-filling mutations that decreased inhibitory activity of alpha(1)-antitrypsin were localized in the loop regions that interact with beta-sheet A distal from the reactive center loop. The results are consistent with the notion that beta-sheet A and the structure around it mobilize when alpha(1)-antitrypsin forms a complex with a target protease.     

A 2.1 A resolution structure of an uncleaved alpha(1)-antitrypsin shows variability of the reactive center and other loops

Serpin (serine protease inhibitor) proteins are involved in diverse physiological processes including inflammation, coagulation, matrix remodeling, and cell differentiation. Deficiency of normal serpin functions leads to various hereditary diseases. Besides their clinical importance, serpin proteins draw much attention due to the large conformational changes that occur upon interaction with proteases. We present here the crystal structure of an uncleaved alpha(1)-antitrypsin determined by the multiple isomorphous replacement method and refined to 2.1 A resolution. The structure, which is the first active serpin structure based on experimental phases, reveals novel conformations in the flexible loops, including the proximal hinge region of the reactive center loop and the surface cavity region in the central beta-sheet, sheet A. The determined loop conformation explains the results of recent mutagenesis studies and provides detailed insights into the protease inhibition mechanism. The high-resolution structure of active alpha(1)-antitrypsin also provides evidence for the existence of localized van-der-Waals strain in the central hydrophobic core.     

Dimers initiate and propagate serine protease inhibitor polymerisation

The serine protease inhibitor (serpin) family can readily form long-chain polymers by a process that underlies a variety of diseases. We show here that monomers of plasma serpins α₁-antitrypsin and antithrombin are stable on incubation with the rate-limiting step in their polymerisation being the formation of the initial dimer. Once formed, the dimers readily interlink to form tetramers and can bind monomers to form trimers and longer oligomers. Cleavage of the only exposed reactive loop, in unit I of the dimers, prevents their interlinkage, but these cleaved dimers can still link to monomers. The rapid binding by the cleaved dimers of a peptide specific to the lower half of β-sheet A of the molecule indicates the ready opening of this β-sheet in unit II of the dimers. The failure of the cleaved dimers to bind peptide-complexed monomers, together with the relative inaccessibility of the P14 hinge residue in the oligomers, is evidence that partial insertion of the reactive loop into its own A-sheet is required for polymer formation. We propose that serpin dimers initiate and propagate polymerisation by having one exposed loop with an optimal conformation as a β-strand donor and a readily opened β-sheet as an acceptor. The sequential reformation of these activated β-interfaces as the oligomer extends, molecule by molecule, provides a model for the fibril and amyloid formation of conformational diseases in general as well as for the infectivity of prion encephalopathies.     

Bypassing the kinetic trap of serpin protein folding by loop extension

The native form of some proteins such as strained plasma serpins (serine protease inhibitors) and the spring-loaded viral membrane fusion proteins are in a metastable state. The metastable native form is thought to be a folding intermediate in which conversion into the most stable state is blocked by a very high kinetic barrier. In an effort to understand how the spontaneous conversion of the metastable native form into the most stable state is prevented, we designed mutations of alpha1-antitrypsin, a prototype serpin, which can bypass the folding barrier. Extending the reactive center loop of alpha1-antitrypsin converts the molecule into a more stable state. Remarkably, a 30-residue loop extension allows conversion into an extremely stable state, which is comparable to the relaxed cleaved form. Biochemical data strongly suggest that the strain release is due to the insertion of the reactive center loop into the major beta-sheet, A sheet, as in the known stable conformations of serpins. Our results clearly show that extending the reactive center loop is sufficient to bypass the folding barrier of alpha1-antitrypsin and suggest that the constrain held by polypeptide connection prevents the conversion of the native form into the lowest energy state.     

Inactivation of human plasma serine proteinase inhibitors (serpins) by limited proteolysis of the reactive site loop with snake venom and bacterial metalloproteinases

Human plasma serine proteinase inhibitors (serpins) gradually lost activity when incubated with catalytic amounts of snake venom or bacterial metalloproteinases. Electrophoretic analyses indicated that antithrombin III, C1-inhibitor, and alpha 2-antiplasmin had been converted by limited proteolysis into modified species which retained inhibitory activity. Further proteolytic attack resulted in the formation of inactivated inhibitors; alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 1-antichymotrypsin were also enzymatically inactivated, but active intermediates were not detected. Sequence analyses indicated that the initial, noninactivating cleavage occurred in the amino-terminal region of the inhibitors. Inactivation resulted in all cases from the limited proteolysis of a single bond near, but not at, the reactive site bond in the carboxy-terminal region of the inhibitors. The results indicate that the serpins have two regions which are susceptible to limited proteolysis--one near the amino-terminal end and another in the exposed reactive site loop of the inhibitor.