Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys14?Cys39 bond into the ovomucoid third domain from silver pheasant

P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹⁴? Cys³⁹ bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹⁴? Cys³⁹ bond into the flexible N‐terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH‐independent hydrolysis constant (K_hyd°) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K_hyd°, but has no effect on the thermal stability. The site‐specific disulfide introduction into the flexible N‐terminal loop of natural Kazal‐type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Structural and functional study of an Anemonia elastase inhibitor, a "nonclassical" Kazal-type inhibitor from Anemonia sulcata

Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized alpha-helical (CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solution structure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by a central alpha-helix and a three-stranded antiparallel beta-sheet of a typical Kazal-type inhibitor such as silver pheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forming a disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3. Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB). Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motif could produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI (AEI analogue) was chemically synthesized when a Cys(4)-Cys(34) bond was changed to a Cys(6)-Cys(31) bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibited almost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structural difference was detected between the two, but the N-terminal loop from Pro(5) to Ile(7) near the putative reactive site (Met(10)-Gln(11)) in the analogue moved by 3.7 A toward the central helix to form the introduced Cys(6)-Cys(31) bond. Such a conformational change in the restricted region correlates with the specificity change of the inhibitor.     

The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites.

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.     

Replacement of P1 Leu18 by Glu18 in the reactive site of turkey ovomucoid third domain converts it into a strong inhibitor of Glu-specific Streptomyces griseus proteinase (GluSGP).

Turkey ovomucoid third domain with P1 Leu18 at its reactive site is an excellent inhibitor of chymotrypsin and elastase and of many other serine proteinases with related specificities. Semisynthetic replacement of P1 Leu18 by Lys18 causes the expected change into a trypsin inhibitor. Strikingly, semisynthetic replacement P1 Leu18 to Glu18 changes turkey ovomucoid third domain into a powerful inhibitor of Glu-specific Streptomyces griseus proteinase, GluSGP. Of the 131 natural avian ovomucoid third domains we have sequenced none have P1 Glu18, but several avian ovomucoid first domains have P1 Glu24. They are weak to moderate inhibitors of GluSGP.     

The covalent structure of the elastase inhibitor from Anemonia sulcata--a "non-classical" Kazal-type protein

The amino-acid sequence of the proteinase inhibitor specific for elastases from the sea anemone Anemonia sulcata was determined from performic-acid oxidized inhibitor and from three cyanogen bromide fragments of reduced and carboxymethylated inhibitor. The molecule consists of a single polypeptide chain formed from 48 amino-acid residues and is stabilized by three intramolecular disulfide bridges. After cyanogen bromide cleavage of the native protein at methionines 10 and 28 followed by chymotryptic cleavage two fragments each containing a single disulfide bridge were isolated. These indicated the location of three intramolecular disulfide linkages between Cys4 and Cys34 (part of A-loop), Cys8 and Cys27 (B-loop) and Cys16 and Cys48 (C-loop). The sequential homology and the disulfide pattern identified the elastase inhibitor as a Kazal-type inhibitor in which, however, not only the CysI-CysII segment is rather short but interestingly the Cys4-Cys34 disulfide anchoring point (i.e. CysI-CysV) in the C-loop is shifted by one turn in the alpha-helical segment towards the C-terminus. Thus, the elastase inhibitor is a non-classical Kazal-type inhibitor with respect to the positioning of the half-cystines. The inhibitor molecule was modelled based on the known three-dimensional structure of the silver pheasant ovomucoid third domain. The shortened amino-terminal segment was arranged in such a manner to allow disulfide bridge formation between the first cysteine Cys4 and the replaced Cys34 under maintenance of a suitable binding loop conformation. The characteristic ovomucoid scaffold consisting of a central alpha-helix, an adjacent three-stranded beta-sheet and the proteinase-binding loop cross-connected through disulfide bridges CysI-CysV and CysIII-CysVI was conserved.     

The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3)

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal-type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a = 4.429 nm, b = 2.115 nm, c = 4.405 nm, beta = 107 degrees. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515-537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen-bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982) Proc. Natl Acad. Sci. USA, 79, 4868-4872].     

Turkey ovomucoid third domain inhibits eight different serine proteinases of varied specificity on the same ...Leu18-Glu19 ... reactive site

We show that eight different serine proteinases--bovine chymotrypsins A and B, porcine pancreatic elastase I, proteinase K, Streptomyces griseus proteinases A and B, and subtilisins BPN' and Carlsberg--interact with turkey ovomucoid third domain at the same Leu18-Glu19 peptide bond, the reactive site of the inhibitor. Turkey ovomucoid third domain was converted to modified (the reactive site peptide bond hydrolyzed) form as documented by sequencing. Complexes of all eight enzymes both with virgin and with modified inhibitor were prepared. All 16 complexes were subjected to kinetically controlled dissociation, and all 16 produced predominantly virgin (greater than 90%) inhibitor, thus proving our point. During this investigation, we found that both alpha-chymotrypsin and especially S. griseus proteinase B convert virgin to modified turkey ovomucoid third domain, even in the pH range 1-2, a much lower pH than we expected. We have also measured rate constants kon and kon* for the association of virgin and modified turkey ovomucoid third domain with several serine proteinases. The kon/kon* ratio is 4.8 X 10(6) for chymotrypsin, but it is only 1.5 for subtilisin Carlsberg. A number of generalizations concerning reactive sites of protein proteinase inhibitor are proposed and discussed.     

Inhibition of neutrophil elastase by recombinant human proteinase inhibitor 9.

Proteinase inhibitor PI9 (PI9) is an intracellular 42-kDa member of the ovalbumin family of serpins that is found primarily in placenta, lung and lymphocytes. PI9 has been shown to be a fast-acting inhibitor of granzyme B in vitro, presumably through the utilization of Glu(340) as the P(1) inhibitory residue in its reactive site loop. In this report, we describe the inhibition of human neutrophil elastase by recombinant human PI9. Inhibition occurred with an overall K(i)' of 221 pM and a second-order association rate constant of 1.5 x 10(5) M(-1) s(-1), indicating that PI9 is a potent inhibitor of this serine proteinase in vitro. In addition, incubation of recombinant PI9 with native neutrophil elastase resulted in the formation of an SDS-resistant 62-kDa complex. Amino-terminal sequence analyses provided evidence that inhibition of elastase occurred through the use of Cys(342) as the reactive P(1) amino acid residue in the PI9 reactive site loop. Thus, PI9 joins its close relatives PI6 and PI8 as having the ability to utilize multiple reactive site loop residues as the inhibitory P(1) residue to expand its inhibitory spectrum.     

Design and characterization of a hybrid miniprotein that specifically inhibits porcine pancreatic elastase

Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.     

Refined X-ray crystal structures of the reactive site modified ovomucoid inhibitor third domains from silver pheasant (OMSVP3*) and from Japanese quail (OMJPQ3*).

Tetragonal and triclinic crystals of two ovomucoid inhibitor third domains from silver pheasant and Japanese quail, modified at their reactive site bonds Met18-Glu19 (OMSVP3*) and Lys18-Asp19 (OMJPQ3*), respectively, were obtained. Their molecular and crystal structures were solved using X-ray data to 2.5 A and 1.55 A by means of Patterson search methods using truncated models of the intact (virgin) inhibitors as search models. Both structures were crystallographically refined to R-values of 0.185 and 0.192, respectively, applying an energy restraint reciprocal space refinement procedure. Both modified inhibitors show large deviations from the intact derivatives only in the proteinase binding loops (Pro14 to Arg21) and in the amino-terminal segments (Leu1 to Val6). In the modified inhibitors the residues immediately adjacent to the cleavage site (in particular P2, P1, P1') are mobile and able to adapt to varying crystal environments. The charged end-groups, i.e. Met18 COO- and Glu19 NH3+ in OMSVP3*, and Lys18 COO- and Asp19 NH3+ in OMJPQ3*, do not form ion pairs with one another. The hydrogen bond connecting the side-chains of Thr17 and Glu19 (i.e. residues on either side of the scissile peptide bond) in OMSVP3 is broken in the modified form, and the hydrogen-bond interactions observed in the intact molecules between the Asn33 side-chain and the carbonyl groups of loop residues P2 and P1' are absent or weak in the modified inhibitors. The reactive site cleavage, however, has little effect on specific interactions within the protein scaffold such as the side-chain hydrogen bond between Asp27 and Tyr31 or the side-chain stacking of Tyr20 and Pro22. The conformational differences in the amino-terminal segment Leu1 to Val6 are explained by their ability to move freely, either to associate with segments of symmetry-related molecules under formation of a four-stranded beta-barrel (OMSVP3* and OMJPQ3) or to bind to surrounding molecules. Together with the results given in the accompanying paper, these findings probably explain why Khyd of small protein inhibitors of serine proteinases is generally found to be so small.     

Spacer Asn determines the fate of Kunitz (STI) inhibitors, as revealed by structural and biochemical studies on WCI mutants

The scaffold of serine protease inhibitors plays a significant role in the process of religation which resists proteolysis of the inhibitor in comparison to a substrate. Although the role of the conserved scaffolding Asn residue was previously implicated in the maintenance of the binding loop conformation of Kunitz (STI) inhibitors, its possible involvement in the prevention of proteolysis is still unexplored. In this paper, we have investigated the specific role of the spacer Asn in the prevention of proteolysis through structural and biochemical studies on the mutants where Asn14 of winged bean chymotrypsin inhibitor (WCI) has been replaced by Gly, Ala, Thr, Leu, and Gln. A residue having no side chain or beta-branching at the 14th position creates deformation and insufficient protrusion of the binding loop, and as a result N14G and N14T lose the ability to recognize proteases. Although the reactive site loop conformation of N14A and N14Q are almost identical to WCI, biochemical results present N14A as a substrate indicating that the methyl group of Ala14 is not suitable to capture the cleaved parts together for religation. The poor inhibitory power of N14L points toward the chemical incompatibility of Leu at the 14th position, although its size is the same as Asn; on the other hand, slight loss of inhibitory potency of N14Q is attributed to the inappropriate placement of the Gln14 polar head, caused by the strained accommodation of its bigger side chain. These observations collectively allow us to conclude that the side chain of spacer Asn fits snugly into the concave space of the reactive site loop cavity and its ND2 atom forms hydrogen bonds with the P2 and P1' carbonyl O at either side of the scissile bond holding the cleaved products together for religation. Through database analysis, we have identified such spacer asparagines in five other families of serine protease inhibitors with a similar disposition of their ND2 atoms, which supports our proposition.     

The crystal structure of guamerin in complex with chymotrypsin and the development of an elastase-specific inhibitor

Guamerin, a canonical serine protease inhibitor from Hirudo nipponia, was identified as an elastase-specific inhibitor and has potential application in various diseases caused by elevated elastase concentration. However, the application of guamerin is limited because it also shows inhibitory activity against other proteases. To improve the selectivity of guamerin as an elastase inhibitor, it is essential to understand the binding mode of the inhibitor to elastase and to other proteases. For this purpose, we determined the crystal structure of guamerin in complex with chymotrypsin at 2.5 Å resolution. The binding mode of guamerin on elastase was explored from the model structure of guamerin/elastase. Guamerin binds to the hydrophobic pocket of the protease in a substrate-like manner using its binding loop. In order to improve the binding selectivity of guamerin to elastase, several residues in the binding loop were mutated and the inhibitory activities of the mutants against elastase and chymotrypsin were monitored. The substitution of the Met36 residue for Ala in the P1 site increased the inhibitory activity against elastase up to 14-fold, while the same mutant showed 7-fold decreased activity against chymotrypsin compared to the wild-type guamerin. Furthermore, the M36A guamerin mutant more effectively protected endothelial cells against cell damage caused by elastase than the wild-type guamerin.     

Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.     

X-ray crystal structure of the complex of human leukocyte elastase (PMN elastase) and the third domain of the turkey ovomucoid inhibitor.

Orthorhombic crystals diffracting beyond 1.7 A resolution, have been grown from the stoichiometric complex formed between human leukocyte elastase (HLE) and the third domain of turkey ovomucoid inhibitor (OMTKY3). The crystal and molecular structure has been determined with the multiple isomorphous replacement technique. The complex has been modeled using the known structure of OMTKY3 and partial sequence information for HLE, and has been refined. The current crystallographic R-value is 0.21 for reflections from 25 to 1.8 A resolution. HLE shows the characteristic polypeptide fold of trypsin-like serine proteinases and consists of 218 amino acid residues. However, several loop segments, mainly arranged around the substrate binding site, have unique conformations. The largest deviations from the other vertebrate proteinases of known spatial structure are around Cys168. The specificity pocket is constricted by Val190, Val216 and Asp226 to preferentially accommodate medium sized hydrophobic amino acids at P1. Seven residues of the OMTKY3-binding segment are in specific contact with HLE. This interaction and geometry around the reactive site are similar as observed in other complexes. It is the first serine proteinase glycoprotein analysed, having two sugar chains attached to Asn159 and to residue 109.     

Specificity and reactive loop length requirements for crmA inhibition of serine proteases

The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (approximately 10(2)-10(4) M(-1)sec(-1)), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (k(diss) approximately 10(-7) sec(-1)), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate (k(cat)/K(M) of approximately 10(6) M(-1) sec(-1)). N-terminal sequencing confirmed that the P1 Arg-P1'Cys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1'Arg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases approximately 10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (10(5)-10(6) M(-1)sec(-1)), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1'Cys-P2'Ala bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites.     

Structural features and molecular evolution of Bowman-Birk protease inhibitors and their potential application

The Bowman-Birk inhibitors (BBIs) are well-studied serine protease inhibitors that are abundant in dicotyledonous and monocotyledonous plants. BBIs from dicots usually have a molecular weight of 8k and are double-headed with two reactive sites, whereas those from monocots can be divided into two classes, one approximately 8 kDa in size with one reactive site (another reactive site was lost) and the other approximately 16 kDa in size with two reactive sites. The reactive site is located at unique exposed surfaces formed by a disulfide-linked β-sheet loop that is highly conserved, rigid and mostly composed of nine residues. The structural features and molecular evolution of inhibitors are described, focusing on the conserved disulfide bridges. The sunflower trypsin inhibitor-1 (SFTI-1), with 14 amino acid residues, is a recently discovered bicyclic inhibitor, and is the most small and potent naturally occurring Bowman-Birk inhibitor.Recently, BBIs have become a hot topic because of their potential applications. BBIs are now used for defense against pathogens and insects in transgenic plants, which has advantages over using toxic and polluting insecticides. BBIs could also be applied in the prevention of cancer, Dengue fever, and inflammatory and allergic disorders, because of their inhibitory activity with respect to the serine proteases that play apivotal role in the development and pathogenesis of these diseases. The canonical nine-residue loop of BBIs/STH-1 provides an ideal template for drug design of specific inhibitors to target their respective proteases.     

Elastase inhibition by the inter-alpha-trypsin inhibitor and derived inhibitors of man and cattle

The inhibitory properties of HI-14 and BI-14, the active 14-kDa parts released from the corresponding human and bovine inter-alpha-trypsin inhibitors, are compared. The structurally homologous inhibitors composed of two tandem Kunitz-type domains differ in their inhibitory specificity, although the reactive site residue in position P1 is occupied by identical (arginine in the C-terminal domain II) or similar (methionine and leucine in the N-terminal domain I of HI-14 and BI-14, respectively) amino-acid residues. The N-terminal domain I of HI-14 is completely inactive against chymotrypsin and pancreatic elastase, whereas BI-14 is a strong inhibitor of these enzymes. Elastase from polymorphonuclear granulocytes interacts with both inhibitors but with different affinities. Compared with the bovine inhibitor, the human inhibitor shows a much lower affinity from this enzyme. Human ITI and its physiological 30-kDa derivative (HI-30) show the same inhibitory properties as HI-14. The differences between human and bovine inhibitors might be explained by a preceding oxidation of Met in vivo of the reactive site residue in position P1 and/or by the influence of the environmental parts connected with this antielastase reactive site region in human ITI or in the active domains thereof.     

The effects of reactive site location on the inhibitory properties of the serpin alpha(1)-antichymotrypsin

The large size of the serpin reactive site loop (RSL) suggests that the role of the RSL in protease inhibition is more complex than that of presenting the reactive site (P1 residue) to the protease. This study examines the effect on inhibition of relocating the reactive site (Leu-358) of the serpin alpha(1)-antichymotrypsin either one residue closer (P2) or further (P1') from the base of the RSL (Glu-342). alpha(1)-Antichymotrypsin variants were produced by mutation within the P4-P2' region; the sequence ITLLSA was changed to ITLSSA to relocate the reactive site to P2 (Leu-357) and to ITITLS to relocate it to P1' (Leu-359). Inhibition of the chymotrypsin-like proteases human chymase and chymotrypsin and the non-target protease human neutrophil elastase (HNE) were analyzed. The P2 variant inhibited chymase and chymotrypsin but not HNE. Relative to P1, interaction at P2 was characterized by greater complex stability, lower inhibition rate constants, and increased stoichiometry of inhibition values. In contrast, the P1' variant inhibited HNE (stoichiometry of inhibition = 4) but not chymase or chymotrypsin. However, inhibition of HNE was by interaction with Ile-357, the P2 residue. The P1' site was recognized by all proteases as a cleavage site. Covalent-complexes resistant to SDS-PAGE were observed in all inhibitory reactions, consistent with the trapping of the protease as a serpin-acyl protease complex. The complete loss in inhibitory activity associated with lengthening the Glu-342-reactive site distance by a single residue and the enhanced stability of complexes associated with shortening this distance by a single residue are compatible with the distorted-protease model of inhibition requiring full insertion of the RSL into the body of the serpin and translocation of the linked protease to the pole opposite from that of encounter.     

Structure of the complex of Streptomyces griseus proteinase B and polypeptide chymotrypsin inhibitor-1 from Russet Burbank potato tubers at 2.1 A resolution

A low molecular weight protein inhibitor of serine proteinases from Russet Burbank potato tubers, polypeptide chymotrypsin inhibitor-1 (PCI-1), has been crystallized in complex with Streptomyces griseus proteinase B (SGPB). The three-dimensional structure of the complex has been solved at 2.1 A resolution by the molecular replacement method and has been refined to a final R-factor (= sigma[[Fo[-[Fc[[/sigma[Fo[) of 0.142 (8.0 to 2.1 A resolution data). The reactive site bond of PCI-1 (Leu38I to Asn39I) is intact in the complex, and there is no significant distortion of the peptide from planarity. The distance between the active site serine O gamma of SGPB and the carbonyl carbon of the scissile bond of PCI-1 is 2.8 A (1 A = 0.1 nm). The inhibitor has little secondary structure, having a three-stranded antiparallel beta-sheet on the side opposite the reactive site and four beta-turns. PCI-1 has four disulphide bridges; these presumably take the place of extensive secondary structure in keeping the reactive site conformationally constrained. The pairing of the cystine residues, which had not been characterized chemically, is as follows: Cys3I to Cys40I, Cys6I to Cys24I, Cys7I to Cys36I, and Cys13I to Cys49I. The molecular structure of SGPB in the PCI-1 complex agrees closely with the structure of SGPB complexed with the third domain of the turkey ovomucoid inhibitor (OMTKY3). A least-squares overlap of all atoms in SGPB gives a root-mean-square difference of 0.37 A. One of the loops of SGPB (Ser35 to Gly40) differs in conformation in the two complexes by more than 2.0 A root-mean-square for the main-chain atoms. Thr39 displays the largest differences with the carbonyl carbon atom deviating by 3.6 A. This conformational alternative is a result of the differences in the molecular structures of the P'4 residues following the reactive site bonds of the two inhibitors. This displacement avoids a close contact (1.3 A) between the carbonyl oxygen of Ser38 of SGPB and Pro42I C beta of PCI-1. The solvent structure of the PCI-1-SGPB complex includes 179 waters, two sulphate or phosphate ions, and one calcium or potassium ion, which appears to play a role in crystal formation. The molecular structure of PCI-1 determined here has allowed the proposal of a model for the structure of a two-domain inhibitor from potatoes and tomatoes, inhibitor II.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutational and immunochemical analysis of plasminogen activator inhibitor 1

We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.