Crystal structures of Aspergillus oryzae aspartic proteinase and its complex with an inhibitor pepstatin at 1.9A resolution

The X-ray structures of Aspergillus oryzae aspartic proteinase (AOAP) and its complex with inhibitor pepstatin have been determined at 1.9A resolution. AOAP was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=49.4A, b=79.4A, and c=93.6A. By the soaking of pepstatin, crystals are transformed into a monoclinic system with the space group C2 and cell dimensions of a=106.8A, b=38.6A, c=78.7A, and beta=120.3 degrees. The structures of AOAP and AOAP/pepstatin complex were refined to an R-factor of 0.177 (R(free)=0.213) and of 0.185 (0.221), respectively. AOAP has a crescent-shaped structure with two lobes (N-lobe and C-lobe) and the deep active site cleft is constructed between them. At the center of the active site cleft, two Asp residues (Asp33 and Asp214) form the active dyad with a hydrogen bonding solvent molecule between them. Pepstatin binds to the active site cleft via hydrogen bonds and hydrophobic interactions with the enzyme. The structures of AOAP and AOAP/pepstatin complex including interactions between the enzyme and pepstatin are very similar to those of other structure-solved aspartic proteinases and their complexes with pepstatin. Generally, aspartic proteinases cleave a peptide bond between hydrophobic amino acid residues, but AOAP can also recognize the Lys/Arg residue as well as hydrophobic amino acid residues, leading to the activation of trypsinogen and chymotrypsinogen. The X-ray structure of AOAP/pepstatin complex and preliminary modeling show two possible sites of recognition for the positively charged groups of Lys/Arg residues around the active site of AOAP.     

Predicting functional residues of the Solanum lycopersicum aspartic protease inhibitor (SLAPI) by combining sequence and structural analysis with molecular docking

The Solanum lycopersicum aspartic protease inhibitor (SLAPI), which belongs to the STI-Kunitz family, is an effective inhibitor of the aspartic proteases human cathepsin D and Saccharomyces proteinase A. However, in contrast with the large number of studies on the inhibition mechanism of the serine proteases by the STI-Kunitz inhibitors, the structural aspects of the inhibition mechanism of aspartic proteases from this family of inhibitors are poorly understood. In the present study, we have combined sequence and structural analysis methods with protein-protein docking to gain a better understanding of the SLAPI inhibition mechanism of the proteinase A. The results suggest that: i) SLAPI loop L9 may be involved in the inhibitor interaction with the proteinase A′s active site, and ii) the residues I144, V148, L149, P151, F152 and R154 are implicated in the difference in the potency shown previously by SLAPI and another STI-Kunitz inhibitor isolated from Solanum tuberosum to inhibit proteinase A. These results will be useful in the design of site directed mutagenesis experiments to understand more thoroughly the aspartic protease inhibition mechanism of SLAPI and other related STI-Kunitz inhibitors.     

X-ray structures of five renin inhibitors bound to saccharopepsin: exploration of active-site specificity

Saccharopepsin is a vacuolar aspartic proteinase involved in activation of a number of hydrolases. The enzyme has great structural homology to mammalian aspartic proteinases including human renin and we have used it as a model system to study the binding of renin inhibitors by X-ray crystallography. Five medium-to-high resolution structures of saccharopepsin complexed with transition-state analogue renin inhibitors were determined. The structure of a cyclic peptide inhibitor (PD-129,541) complexed with the proteinase was solved to 2.5 A resolution. This inhibitor has low affinity for human renin yet binds very tightly to the yeast proteinase (K(i)=4 nM). The high affinity of this inhibitor can be attributed to its bulky cyclic moiety spanning P(2)-P(3)' and other residues that appear to optimally fit the binding sub-sites of the enzyme. Superposition of the saccharopepsin structure on that of renin showed that a movement of the loop 286-301 relative to renin facilitates tighter binding of this inhibitor to saccharopepsin. Our 2.8 A resolution structure of the complex with CP-108,420 shows that its benzimidazole P(3 )replacement retains one of the standard hydrogen bonds that normally involve the inhibitor's main-chain. This suggests a non-peptide lead in overcoming the problem of susceptible peptide bonds in the design of aspartic proteinase inhibitors. CP-72,647 which possesses a basic histidine residue at P(2), has a high affinity for renin (K(i)=5 nM) but proves to be a poor inhibitor for saccharopepsin (K(i)=3.7 microM). This may stem from the fact that the histidine residue would not bind favourably with the predominantly hydrophobic S(2) sub-site of saccharopepsin.     

Key features determining the specificity of aspartic proteinase inhibition by the helix-forming IA3 polypeptide

The 68-residue IA(3) polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2-32 of the polypeptide adopt an amphipathic alpha-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (K(i) < 0.1 nm) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA(3) from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA(3). The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained.     

Catalytic domain structures of MT-SP1/matriptase, a matrix-degrading transmembrane serine proteinase.

The type II transmembrane multidomain serine proteinase MT-SP1/matriptase is highly expressed in many human cancer-derived cell lines and has been implicated in extracellular matrix re-modeling, tumor growth, and metastasis. We have expressed the catalytic domain of MT-SP1 and solved the crystal structures of complexes with benzamidine at 1.3 A and bovine pancreatic trypsin inhibitor at 2.9 A. MT-SP1 exhibits a trypsin-like serine proteinase fold, featuring a unique nine-residue 60-insertion loop that influences interactions with protein substrates. The structure discloses a trypsin-like S1 pocket, a small hydrophobic S2 subsite, and an open negatively charged S4 cavity that favors the binding of basic P3/P4 residues. A complementary charge pattern on the surface opposite the active site cleft suggests a distinct docking of the preceding low density lipoprotein receptor class A domain. The benzamidine crystals possess a freely accessible active site and are hence well suited for soaking small molecules, facilitating the improvement of inhibitors. The crystal structure of the MT-SP1 complex with bovine pancreatic trypsin inhibitor serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MT-SP1, and suggests determinants for the substrate specificity.     

Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis

The view has been widely held that pepsin-like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinformatics search [Rawlings ND & Bateman A (2009) BMC Genomics10, 437] revealed that, in seven of ～ 1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hallmark sequence motifs of pepsin-like aspartic proteinases. The implications of this theoretical observation prompted us to generate biochemical data to validate this finding experimentally. The aspartic proteinase gene from one of the seven identified bacterial species, Shewanella amazonensis, was expressed in Escherichia coli. The recombinant protein, termed shewasin A, was produced in soluble form, purified to homogeneity, and shown to display properties remarkably similar to those of pepsin-like aspartic proteinases. Shewasin A was maximally active at acidic pH values, cleaving a substrate that has been widely used for assessment of the proteolytic activity of other aspartic proteinases, and displayed a clear preference for cleaving peptide bonds between hydrophobic residues in the P1*P1' positions of the substrate. It was completely inhibited by the general inhibitor of aspartic proteinases, pepstatin, and mutation of one of the catalytic Asp residues (in the Asp-Thr-Gly motif of the N-terminal domain) resulted in complete loss of enzymatic activity. It can thus be concluded unequivocally that this Shewanella gene encodes an active pepsin-like aspartic proteinase. It is now beyond doubt that pepsin-like aspartic proteinases are not confined to eukaryotes, but are encoded within some species of bacteria. The distinctions between the bacterial and eukaryotic polypeptides are discussed and their evolutionary relationships are outlined.     

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.     

Crystal structure at 2.6 A resolution of the complex of subtilisin BPN' with streptomyces subtilisin inhibitor

The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN', with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN'. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the "secondary contact region" of SSI seems to be to support the reactive site loop ("primary contact region"). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).     

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R_free = 21.2%) at 1.70 Å resolution and 15.8% (R_free = 19.2%) at 1.85 Å resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly β-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity.     

The structure of a synthetic pepsin inhibitor complexed with endothiapepsin

The conformation of a synthetic polypeptide inhibitor, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 0.20-nm resolution and refined to an agreement factor of 0.20. The inhibitor: Pro Thr Glu Phe-R-Phe Arg Glu (R = -CH2NH-) is based on a chromogenic substrate of pepsin (EC 3.4.23.1). It has, in place of the scissile bond, a reduced peptide group which is resistant to hydrolysis and mimics the tetrahedral transition state. The inhibitor binds in an extended conformation with the reduced bond close to the essential aspartate side-chains of the enzyme. The hydrogen bonds and hydrophobic interactions between the enzyme and the inhibitor do not induce large conformational changes.     

The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae

The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.     

High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme.

The conformation of the synthetic renin inhibitor CP-69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP-69,799 is an oligopeptide transition--state analogue inhibitor that contains a new dipeptide isostere at the P1-P1' position. This dipeptide isostere is a nitrogen analogue of the well-explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1' C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3' pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3'. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.     

Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding

A 1.9-A molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B' and C, the N terminus of helix I, and the beta(4) region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F' and G' helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding.     

A neutron Laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism.

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing nonhydrolyzable analogues of the scissile peptide bond. However, the positions of protons on the catalytic aspartates and the ligand in these complexes have not been determined with certainty. Thus, our objective was to locate crucial protons at the active site of an inhibitor complex since this will have major implications for a detailed understanding of the mechanism of action. We have demonstrated that high-resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1 A to an R-factor of 23.5% and an R(free) of 27.4%. This work represents the largest protein structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exchanged their hydrogen atoms with D2O in the mother liquor. The majority of residues resisting exchange are buried within core beta-sheet regions of the molecule. The neutron maps confirm that the protein has a number of buried ionized carboxylate groups which are likely to give the molecule a net negative charge even at very low pH, thereby accounting for its low pI. The functional groups at the catalytic center have clearly undergone H-D exchange despite being buried by the inhibitor occupying the active site cleft. Most importantly, the data provide convincing evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. This has an important bearing on mechanistic proposals for this class of proteinase.     

Caspase-8 specificity probed at subsite S(4): crystal structure of the caspase-8-Z-DEVD-cho complex

Caspase-8 is an initiator enzyme in the Fas-mediated pathway of which the downstream executioner caspase-3 is a physiological target. Caspases are cysteine proteases that are specific for substrates with an aspartic acid residue at the P(1) position and have an optimal recognition motif that incorporates four amino acid residues N-terminal to the cleavage site. Caspase-8 has been classified as a group III caspase member because it shows a preference for a small hydrophobic residue at the P(4) substrate position. We report the X-ray crystallographic structure of caspase-8 in complex with benzyloxycarbonyl-Asp-Glu-Val-Asp-aldehyde (Z-DEVD), a specific group II caspase inhibitor. The structure shows that the inhibitor interacts favourably with the enzyme in subsite S(4). Kinetic data reveal that Z-DEVD (K(i) 2 nM) is an almost equally potent inhibitor of caspase-8 as the specific group III inhibitor Boc-IETD-aldehyde (K(i) 1 nM). In view of this finding, the original classification of caspases into three specificity groups needs to be modified, at least for caspase-8, which tolerates small hydrophobic residues as well as the acidic residue Asp in subsite S(4). We propose that the subsite S(3) must be considered as an important specificity-determining factor.     

Structural insights into the activation and inhibition of histo-aspartic protease from Plasmodium falciparum

Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 ? resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.     

Prodomain processing of Asp1 (BACE2) is autocatalytic

Generation of the amyloid peptide through proteolytic processing of the amyloid precursor protein by beta- and gamma-secretases is central to the etiology of Alzheimer's disease. The highly elusive beta-secretase was recently identified as a transmembrane aspartic proteinase, Asp2 (BACE). The Asp2 homolog Asp1 (BACE2/DRAP) has also been reported to exhibit beta-secretase cleavage of amyloid precursor protein. Most aspartic proteinases are generated as inactive proenzymes, requiring removal of the prodomain to generate active proteinase. Here we show that prodomain processing of Asp1 occurs between Leu(62) and Ala(63) and is autocatalytic. Asp1 cleaved a maltose-binding protein-Asp1 prodomain fusion protein and a synthetic peptide at this site. Mutation of one of the conserved catalytic aspartic acid residues in the active site of Asp1 to asparagine (D110N) abolished this cleavage. Mutation of P(1)' and P(2)' residues in the substrate to phenylalanine reduced cleavage at this site. Asp1 expressed in cells was the mature form, and prodomain processing occurred intramolecularly within the endoplasmic reticulum/early Golgi. Interestingly, a proportion of mature Asp1 was expressed on the cell surface. When full-length Asp1(D110N) was expressed in COS-7 cells, it was not processed, suggesting that no other proteinase can activate Asp1 in these cells.     

Structure of the carboxypeptidase Y inhibitor IC in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction

Carboxypeptidase Y (CPY) inhibitor, IC, shows no homology to any other known proteinase inhibitors and rather belongs to the phosphatidylethanolamine-binding protein (PEBP) family. We report here on the crystal structure of the IC-CPY complex at 2.7 A resolution. The structure of IC in the complex with CPY consists of one major beta-type domain and a N-terminal helical segment. The structure of the complex contains two binding sites of IC toward CPY, the N-terminal inhibitory reactive site (the primary CPY-binding site) and the secondary CPY-binding site, which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. It was also revealed that IC had the ligand-binding site, which is conserved among PEBPs and the putative binding site of the polar head group of phospholipid. The complex structure and analyses of IC mutants for inhibitory activity and the binding to CPY demonstrate that the N-terminal inhibitory reactive site is essential both for inhibitory function and the complex formation with CPY and that the binding of IC to CPY constitutes a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of IC toward the cognate proteinase provides insights into the inhibitory mechanism of PEBPs toward serine proteinases and into the specific biological functions of IC belonging to the PEBP family as well.     

Reactive site mutants of recombinant protein C inhibitor

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C (APC). The residues F353-R354-S355 (P2-P1-P1′) constitute part of the reactive site loop of PCI with the R-S peptide bond being cleaved by the proteinase. Changing the reactive site P1 and P2 residues to those of either proteinase nexin-1, α₁-proteinase inhibitor or heparin cofactor II resulted in a decrease in inhibitory activity towards thrombin and APC. Changing the P2 residue F353 → P generated a rPCI which was a better thrombin inhibitor, but was 10-fold less active with APC. While these results support the concept that the P1 and P2 residues are important in the specificity of PCI, they suggest that the reactive site residues are not the only determinant of serpin specificity. Kinetic analysis of the rPCI variants was consistent with PCI operating by a mechanism similar to that proposed for other serpins. In this model an intermediary complex forms between inhibitor and proteinase that can proceed to either cleavage of the inhibitor as substrate or formation of an inactive complex.     

Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3

The three-dimensional structures of pepsin inhibitor-3 (PI-3) from Ascaris suum and of the complex between PI-3 and porcine pepsin at 1. 75 A and 2.45 A resolution, respectively, have revealed the mechanism of aspartic protease inhibition by this unique inhibitor. PI-3 has a new fold consisting of two domains, each comprising an antiparallel beta-sheet flanked by an alpha-helix. In the enzyme-inhibitor complex, the N-terminal beta-strand of PI-3 pairs with one strand of the 'active site flap' (residues 70-82) of pepsin, thus forming an eight-stranded beta-sheet that spans the two proteins. PI-3 has a novel mode of inhibition, using its N-terminal residues to occupy and therefore block the first three binding pockets in pepsin for substrate residues C-terminal to the scissile bond (S1'-S3'). The molecular structure of the pepsin-PI-3 complex suggests new avenues for the rational design of proteinaceous aspartic proteinase inhibitors.