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The 0.25-nm X-ray structure of the Bowman-Birk-type inhibitor from mung bean in ternary complex with porcine trypsin
Abstract
The structure of the Bowman-Birk-type inhibitor from mung bean Phaseolus aureus has been determined in ternary complex with porcine trypsin. The complex formed crystals of the trigonal space group P3(1)21 which diffracted to a resolution of 250 pm. Each of the two mung bean protease reactive sites is bound to trypsin according to the standard mechanism for serine proteinase inhibition. The binding loops thereby adopt the canonical conformation for the standard mechanism; however, the sub-van der Waals contact between the active-site serine O gamma (195) and the P1 carbonyl carbon of both loops is significantly smaller (210 pm) than hitherto observed, with continuous electron density connecting the two atoms. The inhibitor is formed by two double-stranded antiparallel beta-sheets, which are connected into a moderately twisted beta-sheet by a network of hydrogen bonds involving main-chain atoms and two water molecules. All contacts with neighbors in the crystal lattice occur between trypsin molecules. This apparently gives rise to an unusual form of disorder where the complexes pack in two orientations Ta:MaMb:Tb and Tb:MbMa:Ta (Ta, Tb = trypsin, Ma = mung bean loop I, Mb = mung bean loop II), such that the asymmetric unit consists of the ternary complex in two orientations, each with half occupancy. This is nearly equivalent to an asymmetric unit which has one trypsin molecule with full occupancy and one mung bean inhibitor with half occupancy and a crystallographic twofold symmetry axis through its center. Because of the approximate twofold symmetry of the inhibitor itself, however, the electron density was interpretable for most of the inhibitor (17 residues at the termini were not resolved) and shows evidence of its double orientation.
... A total of 139,563 reflections (21,466 independent reflections, 98.6% complete to 1.9 Å) were measured with an overall R sym of 0.082. The structure was solved using AMoRe (28), with the trypsin component of the porcine trypsin-mung bean inhibitor complex (29) as starting model. Successive rounds of model building using the program O (30) and refinement using the program X-PLOR (31) parameterized according to Ref. 32 allowed the positioning of all 223 trypsin residues, 31 of LDTI's 46 residues, 149 water molecules, and 1 calcium ion. ...
... Apart from a few surface-located side chains, the trypsin moiety is almost completely defined by electron density. With the exception of the side chain of Tyr 217 , which swings out to accommodate the amino-terminal residues of LDTI ( Fig. 1; see below), no significant conformational changes are seen in the trypsin component compared with other porcine trypsin structures (29,(47)(48)(49). The LDTI moiety is well defined in the vicinity of the proteinase, but it is characterized by elevated temperature factors and disrupted density further away from trypsin. ...
... An indication that the right handed spiral conformation might be well populated in the free inhibitor, and hence of functional significance, comes from the fact that the side chain of trypsin Tyr 217 swings out to accommodate the amino-terminal residues (Fig. 1). In all other published structures of porcine trypsin (29,(47)(48)(49), this side chain makes favorable stacking interactions with the aromatic side chain of Tyr 172 . ...
The x-ray crystal structure of recombinant leech-derived tryptase inhibitor (rLDTI) has been solved to a resolution of 1.9
Å in complex with porcine trypsin. The nonclassical Kazal-type inhibitor exhibits the same overall architecture as that observed
in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase tryptase. The conformation
of the binding region of rLDTI suggests that tryptase has a restricted active site cleft. The basic amino terminus of rLDTI,
apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent
in tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the tryptase molecule.
A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a
role in inhibition of human lung tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch
is in accordance with the known low molecular weight inhibitors of tryptase.
... Three-dimensional structures are available for SBI (Chen et al 1992), PBI (Suzuki et al 1993), the binary complex of trypsin and ABI (Tsunagae et al 1986) and the ternary complex of two trypsins bound to mung bean inhibitor (Lin et al 1993). The coordi nates for the last of these was not available in the Jan'96 release of PDB. ...
... Chymotrypsin coordinates, obtained from the PDB, were rotated such that they superposed onto the trypsin coordinates of the ABI-trypsin complex· The superposition of the inhibitory loop of ABI onto the second inhibitory loop of SBI yielded a transformation which was applied to the rotated chymotrypsin coordinates· These operations resulted in a model of SBI bound to trypsin and chymotrypsin at the two sites (figrue 3)· The lack of extensive short contacts infigure 3 suggests that the two sites can be accessed simultaneously· However, there are a few short contacts involving residues 174 and 95 of trypsin and chymotrypsin respectively· These short contacts can be relieved so as to enable formation of hydrogen bonds that might stabilize the ternary complex· Such hydrogen bonds have been reported to exist in the crystal structure of due to the different disposition of the active site loops in SBI and PBI with respect to their conserved cores. the complex of mung bean BBI with two trypsin molecules (Lin et al 1993). The inhibitory loops adopt different spatial conformations when the structures of SBI and PBI are superposed· Therefore, a similar superposition was carried out to obtain a ternary complex of trypsin, chymotrypsin and PBI· In this model the number of short contacts were less (figure 3)· ...
... The reactive site loops deviate appreciably from the two-fold symmetry relating the core (figure 1)· Models of trypsin and chymotrypsin bound to BBI were constructed based on the known structure of ABI inhibitor-trypsin complex.Figure 3 show that there are minor short contacts concentrated mainly around the residues 174 of trypsin and 95 of chymotrypsin. The crystal structure of the mung bean inhibitor complex showed that the two trypsin molecules interact by means of hydrogen bonds between the residues 174 of one trypsin and 95 of the other (Lin et al 1993). In our model, most of the atoms involved in the short contacts are separated by a distance of about 2·0 A. About 10 atoms have distances less than 2·0A. ...
Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These
inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of
the known three-dimensional structures of BBIs revealed similarities and subtle differences. The hydrophobic core, deduced
from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed
BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold
symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid
core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric
soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting
near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated
with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI,
binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor
were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.
... The so-called binding loop fits into the convex active site of the target protease, in a substrate-like manner [15,24]; the resulting non-covalent complex renders the protease inactive. The double-headed BBI have a characteristic highly conserved array of disulphide bridges occurring between the 14 cysteine residues [25][26] Fig. (2). The seven disulphide bonds are arranged in the following pairs: C1-C14, C2-C6, C3-C13, C4-C5, C7-C9, C8-C12 and C10-C11. ...
... Marked differences were shown for the inhibition of trypsin (K i values in the range from 9 nM to >1mM, depending on the nature of the P 2 ' amino acid), with Ile being the best residue at this position and having the lowest K i value. The residues Ser and Pro at positions P 1 ' and P 3 ', respectively, are strictly conserved in both binding loops of BBI and are believed to be involved in an intramolecular hydrogen bonding network within the loops [26]. However, through NMR analyses, it has been suggested that Ser at P 1 ' is not essential for the integrity of the binding loop and it makes a small contribution to trypsin affinity, but none to stability against proteolytic turnover [31]. ...
Protease inhibitors (PI) have long been considered to be antinutritional compounds in legume crops due to their ability to decrease the protein digestibility and adsorption of dietary proteins. In recent years, renewed interest in PI has followed the recognition that certain plant PI are effective at preventing or suppressing carcinogenic processes in a wide variety of in vitro and in vivo animal model systems (Kennedy, 1998). These reports suggest a positive contribution of PI to the nutritional value of dietary proteins from vegetable sources. The selection of plant PI for plant breeding programs aimed at human health depends on elucidation of the molecular basis for variation in their biological activity. Distinct PI have been identified in legumes with many seed inhibitors inhibiting trypsin and chymotrypsin at distinct sites (Figure 1). The significance of the two active sites is likely to be different in terms of their exploitation. The trypsin inhibitory site has been implicated in the negative effect on bioavailability of dietary proteins in food and feed (Liener, 1994) and in the protection of plants against insects (Volpicella et al., 2000) and fungi (Giudici et al., 2000). The chymotrypsin inhibitory site has been implicated in the anticarcinogenic effect of the soy-derived Bowman-Birk inhibitor (BBI) (Kennedy et. al., 1993). We are investigating the properties of a range of PI homologous to BBI that include sequence variants as well as variants that arise from post-translational processing. We have expressed two pea variant PI and their corresponding C-terminally processed forms as recombinant proteins and are assessing the antiproliferative properties of the variants on colon cancer cells using an in vitro cell assay system designed for high-throughput and rapid screening.
... Kunitz-type Serine Three-fold -trefoil 1 [3] Kunitz-type Serine Three-fold -trefoil 0 [134] [135] Monocot BBI Serine Two antiparallel –sheet (Knottin) 5 [136] [137] [138] Dicot BBI Serine Two antiparallel –sheet (Knottin) 7 [136] [137] [138] Potato type I Serine Four parallel and anti-parallel -sheet against an -helix 0 [139] Potato type II Serine Triple anti-parallel -sheet joined by long loops and a single turn of a 310-helix 4 [140] [141] [142] Cystatin Cysteine Five anti-parallel -sheet around a five turn -helix 0 [143] [144] PCI Metallo Cystine-knot 3 [145] ily. Regardless, Potato type I (Pin I) and type II (Pin II) have similar conformations in the loops which bind to the proteases [23] [24], their secondary structures differ (Table 1). ...
... Kunitz-type Serine Three-fold -trefoil 1 [3] Kunitz-type Serine Three-fold -trefoil 0 [134] [135] Monocot BBI Serine Two antiparallel –sheet (Knottin) 5 [136] [137] [138] Dicot BBI Serine Two antiparallel –sheet (Knottin) 7 [136] [137] [138] Potato type I Serine Four parallel and anti-parallel -sheet against an -helix 0 [139] Potato type II Serine Triple anti-parallel -sheet joined by long loops and a single turn of a 310-helix 4 [140] [141] [142] Cystatin Cysteine Five anti-parallel -sheet around a five turn -helix 0 [143] [144] PCI Metallo Cystine-knot 3 [145] ily. Regardless, Potato type I (Pin I) and type II (Pin II) have similar conformations in the loops which bind to the proteases [23] [24], their secondary structures differ (Table 1). ...
The overwhelming demand for food requires the application of technology on field. An important issue that limits the productivity of crops is related to insect attacks. Hence, several studies have evaluated the application of different compounds to reduce the field losses, especially insecticide compounds from plant sources. Among them, plant protease inhibitors (PIs) have been studied in both basic and applied researches, displaying positive results in control of some insects. However, certain species are able to bypass the insecticide effects exerted by PIs. In this review, we disclosed the adaptive mechanisms showed by lepidopteran and coleopteran insects, the most expressive insect orders related to crop predation. The structural aspects involved in adaptation mechanisms are presented as well as the newest alternatives for pest control. The application of biotechnological tools in crop protection will be mandatory in agriculture, and it will be up to researchers to find the best candidates for effective control in long-term.
... Variation was introduced into the library at selected positions in order to generate diversity likely to modify specificity. Interactions with the proteinase are expected to be exclusively via the`upperface' of the loop, which spans the region P 4 ±P 2 H [35]. Ser is highly conserved at the P 1 H position in BBI proteins [36], and for this reason this residue was not randomised. Similarly, Thr at P 2 shows a high degree of conservation [36]. ...
... Further analysis of all included amino acids at this locus has confirmed Thr as the optimal residue [36]. In the crystal structures of both BBI±trypsin [35] and OMTKY3±HLE complex [26] (where a Thr residue is also found at P 2 ), the P 2 Thr is found to make close contact with His57. Further, in the structure of BBI complexed with trypsin [37], the hydroxyl function of this Thr is involved in hydrogen bonds within the loop structure. ...
A resin-bound cyclic peptide library was constructed based on the sequence of the reactive-site loop of Bowman–Birk inhibitor, a proteinase inhibitor protein. The constrained loop sequence, which incorporates the minimal proteinase-binding motif, was retained throughout the library, but selected residues known to be important for inhibitor specificity were randomised. The approach was used to create a ‘one bead, one peptide’ library with 8000 variants resulting from randomization at three target locations in the sequence (P4, P1 and P2′). This library allows us to examine the degree to which variations in this proteinase-binding motif can redirect activity, as well as providing information about the binding specificity of a proteinase target. Screening this library for binding to human leucocyte elastase identified sequences with a strong consensus, and on resynthesis all were found to act as inhibitors, with Ki values as low as 65 nm. Human leucocyte elastase is known to have a substrate preference for small alkyl chains at the P1 locus, with valine being preferred. However, alanine and not the expected valine was found in 21 out of 23 identified sequences. The remaining two sequences had threonine at P1, a finding that would be hard to predict based on substrate specificity alone. Further analysis of resynthesized peptides demonstrated that valine substitution results in an analogue that is hydrolysed far more rapidly than ones having library-selected P1 residues. Testing of the human leucocyte elastase-selected sequences as inhibitors of porcine pancreatic elastase demonstrates a significant difference in the specificity of the P4 locus between these two proteinases.
... Each head is made of a tricyclic domain in which the functional reactive site loop is located. Loop I typically inhibits trypsin and loop I' inhibits chymotrypsin (Chen et al., 1992;Lin et al., 1993;Voss et al., 1996;Werner and Wemmer, 1992). The interaction of Bowman-Birk inhibitor with serine proteases occurs via a welldefined disulfide linked short beta-sheet region which generates a non-covalent complex that renders the serine protease inactive (McBride and . ...
... The stability and consequently the biological activity of the Bowman-Birk inhibitorbased peptides are supported by an extensive intramolecular hydrogen-bond network. The main-chain to main-chain hydrogen bond between P2 and P1′ (Fig. 2, dash lines) is responsible for projecting the P1 side-chain outwards for the primary interaction with the enzyme (Lin et al., 1993;Voss et al., 1996). The cis-Pro-trans-Pro motif in the P3′-P4′ positions and, the disulphide bridge covalently linking the P3-P6′ positions together with the hydrogen bond network guarantees the β-hairpin conformation typical of the Bowman-Birk inhibitor protein (Brauer et al., 2001;Mcbride et al., 1999;McBride et al., 2002). ...
Chronic wounds are the result of excessive amounts of tissue destructive proteases such as human neutrophil elastase (HNE). The high levels of this enzyme found on those types of wounds inactivate the endogenous inhibitor barrier thus, the search for new HNE inhibitors is required. This work presents two new HNE inhibitor peptides, which were synthesized based on the reactive-site loop of the Bowman-Birk inhibitor protein. The results obtained indicated that these new peptides are competitive inhibitors for HNE and, the inhibitory activity can be modulated by modifications introduced at the N- and C-terminal of the peptides. Furthermore, these peptides were also able to inhibit elastase from a human wound exudate while showing no cytotoxicity against human skin fibroblasts in vitro, greatly supporting their potential application in chronic wound treatment.
... The so-called binding loop fits into the convex active site of the target protease, in a substrate-like manner [15,24]; the resulting non-covalent complex renders the protease inactive. The double-headed BBI have a characteristic highly conserved array of disulphide bridges occurring between the 14 cysteine residues [25][26] Fig. (2). The seven disulphide bonds are arranged in the following pairs: C1-C14, C2-C6, C3-C13, C4-C5, C7-C9, C8-C12 and C10-C11. ...
... Marked differences were shown for the inhibition of trypsin (K i values in the range from 9 nM to >1mM, depending on the nature of the P 2 ' amino acid), with Ile being the best residue at this position and having the lowest K i value. The residues Ser and Pro at positions P 1 ' and P 3 ', respectively, are strictly conserved in both binding loops of BBI and are believed to be involved in an intramolecular hydrogen bonding network within the loops [26]. However, through NMR analyses, it has been suggested that Ser at P 1 ' is not essential for the integrity of the binding loop and it makes a small contribution to trypsin affinity, but none to stability against proteolytic turnover [31]. ...
Bowman-Birk inhibitors and their variants (BBI) from legumes, such as soybean, pea, lentil and chickpea, are a class of naturally-occurring protease inhibitors which have potential health-promoting properties within the gastrointestinal tract. BBI can resist both acidic conditions and the action of proteolytic enzymes, and transit through the stomach and small intestine without major degradation, permitting significant amounts to reach the large intestine in active form to exert their reported anti-carcinogenic and anti-inflammatory properties. These potential pharmacological benefits have been linked recently to the intrinsic ability of BBI to inhibit serine proteases, and the data suggest that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI. However, the therapeutic targets and the action mechanisms of BBI remain unknown. Their elucidation will provide insights into the properties of these plant protease inhibitors as colorectal chemopreventive agents, providing a strong base for the development of legume crops and their products as pro-nutritional, health-promoting food. The deployment of modern genomic tools and genome sequence information are underpinning studies of natural and induced polymorphism in BBI. Genetic markers for BBI variants with improved properties can be exploited ultimately in legume breeding programmes to assist the introgression of such variant genes and the development of superior genotypes for human nutrition.
... There are five disulfide bonds present in each inhibitor. These inhibitors can be grouped into a novel family of Ascaris inhibitors, as they display no sequence identity with [28][29][30], squash seed trypsin inhibitor (squash seed inhibitor family; [31]), serpins [32][33][34][35][36][37][38] and hirudin [39,40]. The inhibitors from these different families have a common conformation for the reactive-site loop, while displaying completely different overall structures (see [41,42] for reviews). ...
... The other (Pro42 I to Arg44 I) is between -strands 3 and 4. Interestingly, -structures are more commonly observed than a-helices among protein inhibitors of serine proteinases. While there are no all-at structures, there are all-3 structures, such as Erythina trypsin inhibitor [19], mung bean trypsin inhibitor [30], mucus proteinase inhibitor (SLPI) [27] and squash seed trypsin inhibitor-1 (CMTI-I) [31]. ...
The intestinal parasitic worm, Ascaris suum, produces a variety of protein inhibitors that defend the organism against the host's proteinases. Eight different proteins from Ascaris suum have been identified as inhibitors of serine proteinases, targeting chymotrypsin, elastase and trypsin. These inhibitors share 30-40% sequence identity with one another, but have virtually no sequence identity with members of any of the other families of serine proteinase inhibitors.
The crystal structure of the complex of porcine pancreatic elastase with a chymotrypsin/elastase inhibitor from Ascaris suum (the C/E-1 inhibitor) has been solved to 2.4 A resolution by the molecular replacement method. The C/E-1 inhibitor exhibits a novel folding motif. There are only two small beta-sheets and two single-turn 3(10)-helices in this inhibitor. Unlike the majority of proteins, the C/E-1 inhibitor does not have a hydrophobic core. The presence and unique topography of the five disulfide bridges suggests that they play important roles in maintaining the tertiary structure of the inhibitor. In addition, the side chains of several charged residues from electrostatic and hydrogen-bonding cascades, which also probably compensate for the lack of extensive secondary structures and a hydrophobic core. The reactive-site loop of this inhibitor displays a conformation that is characteristic of most serine proteinase inhibitors.
The structure of the C/E-1 inhibitor confirms that inhibitors from Ascaris suum belong to a novel family of proteinase inhibitors. It also provides conclusive evidence for the correct disulfide bridge connections. The C/E-1 inhibitor probably acts by a common inhibitory mechanism proposed for other substrate-like protein inhibitors of serine proteinases. The unusual molecular scaffolding presents a challenge to current folding algorithms. Proteins like the C/E-1 inhibitor may provide a valuable model system to study how the primary sequence of a protein dictates its three-dimensional structure.
... In a similar approach, Lopes et al. (2004) aligned all available trypsin sequences characterized as sensitive or insensitive to Kunitztype inhibitor (Bown et al., 1997;Mazumdar-Leighton and Broadway, 2001) with porcine trypsin. After discounting conserved positions and positions not typical of sensitive or insensitive trypsin, the remaining positions that agree with those involved in porcine trypsin-PI (Bowman-Birk type, Lin et al., 1993;Kunitz type, Song and Suh, 1998) or substrate (Koepke et al., 2000) interactions were: 60,94,97,98,99,188,190,213,215,217,219,228. These positions support the tree branches in a neighbour-joining analysis of sensitive (I, III) and insensitive (II) trypsin sequences (Lopes et al., 2004). ...
In this chapter, we further explore the biochemical adaptations of insects to avoid the inhibitors of endopeptidase, α-amylase, polygalacturonase, and lipase inhibitors. How herbivorous insects deal with the proteinaceous inhibitors of digestive enzymes found in host plants is a key question in the evolution and in the ecology of insect-plant interactions. Those types of inhibitors are not the only ones found in plants, but they are by far the most abundant and for which there is more data in the literature. We will begin by discussing adaptations to endopeptidase inhibitors, which are the best-known and most diverse. We will then turn to carbohydrate digestion inhibitors, particularly how insects adapt to the ingestion of α-amylase, polygalacturonase, and lipase inhibitors.
... et al. (2004) aligned all available trypsin sequences characterized as sensitive or insensitive to Kunitz-type inhibitor ( Bown et al. , 1997;Mazumdar-Leighton and Broadway, 2001a ) with porcine trypsin. After discounting conserved positions and positions not typical of sensitive or insensitive trypsins, the remaining positions that agreed with those involved in porcine trypsin -PI (Bowman-Birk type, Lin et al. , 1993 ;Kunitz type, Song and Suh, 1998 ) or substrate ( Koepke et al. , 2000 ) interactions were: 60,94,97,98,99,188,190,213,215,217,219,228. Th ese positions support the tree branches in a neighbor-joining analysis of sensitive (I, III) and insensitive (II) trypsin sequences ( Lopes et al. , 2004 ). ...
... The reactive sites in these domains are usually specific to trypsin, chymotrypsin, and elastase [37]. In addition, seven conserved disulfide bonds are present in the active site configurations that also act as stabiliser to these inhibitors [38]. In monocotyledonous plants, two types of BBI have been identified. ...
Bowman-Birk Inhibitor (BBI) is one of the subfamilies of serine protease inhibitors. Numerousstudies have shown that in plants, BBI functions as part of their defense mechanism againstpathogens and microorganisms. The BBI is also known to have anti-carcinogenic properties.Furthermore, the BBI has been reported to function in controlling abiotic stresses such assalinity and drought stresses.. Abiotic stresses are the major problems in agricultural industry.Therefore, numerous researches have been carried out to characterize the BBI and to determineits roles during biotic and abiotic stresses. This paper presents a review regarding therelationship between Bowman-Birk inhibitor and the plant defensive mechanism against abioticstresses.
... The actions of soybean inhibitors were reported to be similar in rat and humans on trypsin and chymotrypsin [129]. According to Lin et al. [136], Bowman-Birk inhibitors inhibit Fig. 4 An example of saponin. Here is Bacopa saponins C with its carbohydrate moiety and its steroidal moiety trypsin via binding with lysine or arginine at the P1 residue. ...
... The first reactive site in these inhibitors is usually specific for trypsin, chymotrypsin and elastase (Qi et al. 2005). The active site configuration in these inhibitors is stabilized by the presence of seven conserved disulfide bonds (Chen et al. 1992;Lin et al. 1993). ...
Enzymes hydrolysing peptide bonds have some overlapping terms, these include, proteases, proteinases and peptidases (Barrett et al. 1998). The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB 1992) recommended peptidase as the general term for enzymes hydrolyzing peptide bonds, which is further divided into exopeptidases, which catalyse the cleavage of one or a few amino acids from N-/C-terminus, and endopeptidases, which cleave the internal peptide bonds of polypeptides. The term “protease” includes both exopeptidases and endopeptidases while “proteinase” designates only endopeptidases (Barrett et al. 1998). Proteolytic enzymes are extensively found in plants, animals and in microorganisms (Kenny 1999) with a major role involved in every aspect of their physiology and development. Proteases are highly specific to their substrate, and the specificity depends on the localization of the substrate and the proteolytic enzyme, and structural and chemical properties at the active site of the enzyme
... The actions of soybean inhibitors were reported to be similar in rat and humans on trypsin and chymotrypsin [129]. According to Lin et al. [136], Bowman-Birk inhibitors inhibit Fig. 4 An example of saponin. Here is Bacopa saponins C with its carbohydrate moiety and its steroidal moiety trypsin via binding with lysine or arginine at the P1 residue. ...
... The first reactive site in these inhibitors is usually specific for trypsin, chymotrypsin and elastase (Qi et al. 2005). The active site configuration in these inhibitors is stabilized by the presence of seven conserved disulfide bonds (Chen et al. 1992;Lin et al. 1993). ...
Enzymes hydrolysing peptide bonds have some overlapping terms, these include, proteases, proteinases and peptidases (Barrett et al. 1998). The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB 1992) recommended peptidase as the general term for enzymes hydrolyzing peptide bonds, which is further divided into exopeptidases, which catalyse the cleavage of one or a few amino acids from N-/C-terminus, and endopeptidases, which cleave the internal peptide bonds of polypeptides. The term “protease” includes both exopeptidases and endopeptidases while “proteinase” designates only endopeptidases (Barrett et al. 1998). Proteolytic enzymes are extensively found in plants, animals and in microorganisms (Kenny 1999) with a major role involved in every aspect of their physiology and development. Proteases are highly specific to their substrate, and the specificity depends on the localization of the substrate and the proteolytic enzyme, and structural and chemical properties at the active site of the enzyme.Their mode of action varies among all families and groups of proteases. Some of them work individually, some work in cascades in cooperation with other proteases and some form complexes constituting an active proteolytic machine. In plants, various roles of proteolytic enzymes involves: removal of misfolded, modified, and/or mistargeted proteins; supply of amino acids during translation; maturation of zymogens and peptide hormones by partial cleavages; control of metabolism and homeostasis by altering the levels of key enzymes and regulatory proteins; and the cleavage of targeted signals from proteins prior to their final integration into organelles (Vierstra 1996). In insects, proteolysis allows digestion of wide range of food diet mediated by concerted action of several proteases and several of them such as trypsin, chymotrypsin, aminopeptidase, etc., have been characterized from a vast variety of insect pests till now (Anwar and Saleemuddin 2002; Sanatan et al. 2013; Akbar et al. 2017). The insect attack on plants triggers the production of a series of secondary metabolites; definsins, thionines, lectins, and protease inhibitors which altogether constitute the defensive armoury of plants (Buchmanan et al. 2002). Plant protease inhibitors are proteinacious in nature and inhibit insect gut proteases by binding tightly to the active site, forming an essentially irreversible complex. The inability to utilize ingested protein and to recycle digestive enzymes results in critical amino acid deficiency, which affects the growth, development and survival of the herbivore (Chougule et al. 2008). In this chapter, we aim to summarize the interactions between insect midgut proteases and the plant protease inhibitors induced as a result of insect attack.
... The presence of an element forcing a peptide bond into a cis conformation in the P 3 ' position has been evaluated as a crucial element for the bioactivity of canonical inhibitors because the canonical conformation strictly requires a cis amide bond in this position. [23,133] To inspect the effect of the presence or absence of a cis conformation in the P 3 ' position for the present small molecule analogs of Ahp cyclodepsipeptides, two different analogs were investigated. ...
Im Rahmen dieser Dissertation sollten Naturstoffe und davon abgeleitete Derivate synthetisiert und im Hinblick auf ihre biologische Aktivität als Protease-Inhibitoren untersucht werden.
Für die Naturstoffklasse der 3-Amino-6-Hydroxy-2-piperidon(Ahp)-Cyclodepsipeptide, die als nicht-kovalente Serin-Protease-Inhibitoren bekannt sind, konnte eine Festphasensynthese basierend auf einem allgemeinen Ahp-Vorläufermolekül entwickelt werden. Für den Ahp-Vorläufer wurde eine Lösungssynthese entwickelt. Die Festphasenstrategie ermöglicht die Synthese maßgeschneiderter Ahp-Cyclodepsipeptide; sie beinhaltet eine Veresterung an der festen Phase, sowie eine Festphasen-Macrolaktamisierung und etabliert ein neues Protokoll zur Generierung eines Aldehyds bei Abspaltung von der festen Phase. Zur Überprüfung der neu-entwickelten Strategie wurde der Chymotrypsin-Inhibitor Symplocamide A erfolgreich an der festen Phase synthetisiert und im Anschluss auf seine biologische Aktivität hin untersucht.
Mit Symplocamide A als der Leitstruktur wurden vereinfachte Analoga von Ahp-Cyclodepsipeptiden synthetisiert, um zu überprüfen ob diese Analoga, wie die Ahp-Cyclodepsipeptide auch, die sogenannte kanonische Konformation einnehmen. Die kanonische Konformation wurde zuerst in proteinogenen Serinprotease-Inhibitoren beobachtet und studiert. Kürzlich wurden auch peptidische Analoga dieser Inhibitoren untersucht und ein Zusammenhang mit den Ahp-Cyclodepsipeptiden erkannt, welche ebenfalls durch die Einnahme der kanonischen Konformation inhibieren.
Im Rahmen dieser Dissertation war es möglich zu zeigen, dass die Ahp-Einheit unter Retention der biologischen Aktivität durch kommerziell erhältliche Aminosäuren ersetzt werden kann. Mittels Struktur-Wirkungsuntersuchungen konnten zudem die kritischen Determinanten zur Einnahme der kanonischen Konformation bestimmt, sowie ein Einblick in die molekulare Grundlage einer effizienten Inhibition gewonnen werden.
Für den Naturstoff Symplostatin 4 wurde eine konvergente Lösungssynthese entwickelt, die einen flexiblen Zugang zu modifizierten Derivaten ermöglichte. Speziell die Synthese von Sonden für die Untersuchung von Symplostatin 4 mittels aktivitätsbasiertem Proteinprofiling war durch eine 1,3-dipolare Huisgen-Cycloaddition von propargyl-modifizierten Spezies mit Azid-modifizierten Reportermolekülen vereinfacht möglich.
Durch aktivitätsbasiertes Profiling konnten mit den Cystein-Proteasen RD21A und RD21B die Zielenzyme von Symplostatin 4 in der Modellpflanze Arabidopsis thaliana identifiziert werden.
Desweiteren wurde die anti-Malaria-Aktivität, die für das chemisch identische Gallinamide A publiziert worden war, für Symplostatin 4 und seine Derivate studiert. Im Rahmen von Untersuchungen mit dem Malariaerreger Plasmodium falciparum konnte festgestellt werden, dass Symplostatin 4 ein nanomolarer Inhibitor der Cystein-Proteasen Falcipain 2 und 3 ist, die für den Hemoglobinverdau zuständig sind und als wichtige Zielenzyme einer alternativen Malaria-Chemotherapie betrachtet werden.
... In a similar approach, 10011-GILBERT-9780123847478 Lopes et al. (2004) aligned all available trypsin sequences characterized as sensitive or insensitive to Kunitz-type inhibitor ( Bown et al. , 1997;Mazumdar-Leighton and Broadway, 2001a ) with porcine trypsin. After discounting conserved positions and positions not typical of sensitive or insensitive trypsins, the remaining positions that agreed with those involved in porcine trypsin -PI (Bowman-Birk type, Lin et al. , 1993 ;Kunitz type, Song and Suh, 1998 ) or substrate ( Koepke et al. , 2000 ) interactions were: 60, 94,97,98,99,188,190,213,215,217,219,228. Th ese positions support the tree branches in a neighbor-joining analysis of sensitive (I, III) and insensitive (II) trypsin sequences ( Lopes et al. , 2004 ). ...
... et al. (2004) aligned all available trypsin sequences characterized as sensitive or insensitive to Kunitz-type inhibitor ( Bown et al. , 1997;Mazumdar-Leighton and Broadway, 2001a ) with porcine trypsin. After discounting conserved positions and positions not typical of sensitive or insensitive trypsins, the remaining positions that agreed with those involved in porcine trypsin -PI (Bowman-Birk type, Lin et al. , 1993 ;Kunitz type, Song and Suh, 1998 ) or substrate ( Koepke et al. , 2000 ) interactions were: 60,94,97,98,99,188,190,213,215,217,219,228. Th ese positions support the tree branches in a neighbor-joining analysis of sensitive (I, III) and insensitive (II) trypsin sequences ( Lopes et al. , 2004 ). ...
The aim of this chapter is to review the recent and spectacular progress in the study of insect digestive biochemistry. It tries to establish uniform parameters for studying insect digestive enzymes, providing an overview of the biochemistry of insect digestion, and discusses factors affecting digestive enzymes in vivo. It reviews digestive enzymes and microvillar proteins, with the emphasis on molecular aspects. Further it describes the details of the digestive biochemical process alongside insect evolution and discusses data on digestive enzyme secretion mechanisms. Digestion is the process by which food molecules are broken down into smaller molecules that are absorbed by cells in the gut tissue. This process is controlled by digestive enzymes and is dependent on their localization in the insect gut. Enzyme kinetic parameters are meaningless unless assays are performed in conditions in which enzymes are stable. If researchers adopt uniform parameters and methods, comparisons among similar and different insect species will be more meaningful.
... The first reactive site in these inhibitors is usually specific for trypsin, chymotrypsin, and elastase (Qi et al., 2005). The active site configuration in these inhibitors is stabilized by the presence of seven conserved disulfide bonds (Chen et al., 1992;Lin et al., 1993). In the case of double-headed BBIs, it has been found that the relative affinity of binding of proteases is altered when one site is already occupied. ...
... In the pro tease binding loop, serine and proline are highly con served residues [42]. Serine residues may support intramolecular hydrogen bonding in the inhibitor loop [43], and proline residues could be have inhibitory role and stabilize cis conformation [44]. These data are in agreement with our findings. ...
Protease inhibitors (PIs) are generally small proteins that have been identified in plants. The wip1 gene codes for wound induced protein, which is similar to serine PIs of the Bowman–Birk family (BBIs). In this study, we analyzed 10 wip1 genes of Turkish maize varieties to understand the structure and characteristics of the wip1 genes and proteins in maize. We found that genetic variability of wip1genes was higher (π: 0.0173) than reported in previous studies. Tajima’s D value was found to be positive (1.73), suggesting overdominant selection in these loci. According to phylogenetic analysis of wip1 proteins, monocot and dicot BBIs were separated independently, and Turkish varieties were clustered with each other generally. The 3D structures of wip1 proteins indicated that several wip1 proteins had structural divergence in active loops, containing various numbers of cysteine residues ranging between 7 and 9. Particularly, Cys74 was identified in Kocbey and Gozdem varieties, whereas Cys98 was only in the Gozdem variety. Also, a critical serine residue (Ser98) was observed in two varieties – Antbey and Batem Efe. These results can contribute to understanding the role of wip1 genes and corresponding proteins in maize.
... The first reactive site in these inhibitors is usually specific for trypsin, chymotrypsin and elastase (Qi et al. 2005). The active site configuration in these inhibitors is stabilized by the presence of seven conserved disulfide bonds (Chen et al. 1992;Lin et al. 1993). On the other hand BBIs from monocotyledonous plants are of two types. ...
Plant serine protease inhibitors are defense proteins crafted by nature for inhibiting serine proteases. Use of eco-friendly, sustainable and effective protein molecules which could halt or slow down metabolism of nutrients in pest would be a pragmatic approach in insect pest management of crops. The host-pest complexes that we observe in nature are evolutionary dynamic and inter-depend on other defense mechanisms and interactions of other pests or more generally speaking symbionts with the same host. Insects have co-evolved and adapted simultaneously, which makes it necessary to investigate serine protease inhibitors in non-host plants. Such novel serine protease inhibitors are versatile candidates with vast potential to overcome the host inhibitor-insensitive proteases. In a nutshell exploring and crafting plant serine proteinase inhibitors (PIs) for controlling pests effectively must go on. Non-host PI seems to be a better choice for coevolved insensitive proteases. Transgenic plants expressing wound inducible chimaeric PIs may be an outstanding approach to check wide spectrum of gut proteinases and overcome the phenomenon of resistance development. Thus, this article focuses on an entire array of plant serine protease inhibitors that have been explored in the past decade, their mode of action and biological implications as well as applications.
... The overall stretch of 30 residues forms two domains, each with an independent RSL. The N-terminal domain has four disulfide bridges, whereas the C-terminal domain has only three [30][31][32]. The absence of a hydrophobic core in the bowtie motif is compensated by coordination of selfassociations such as homodimers, trimers or more complex aggregations through hydrophobic surface patches, which are further stabilized by hydrophilic clusters [33]. ...
... The first reactive site in these inhibitors is usually specific for trypsin, chymotrypsin and elastase (Qi et al., 2005). The activesite configuration in these inhibitors is stabilized by the presence of seven conserved disulfide bonds (Chen et al., 1992;Lin et al., 1993). ...
Proteases, though essentially indispensable to the maintenance and survival of their host organisms, can be potentially damaging when overexpressed or present in higher concentrations, and their activities need to be correctly regulated. An important means of regulation involves modulation of their activities through interaction with substances, mostly proteins, called protease inhibitors. Some insects and many of the phytopathogenic microorganisms secrete extracellular enzymes and, in particular, enzymes causing proteolytic digestion of proteins, which play important roles in pathogenesis. Plants, however, have also developed mechanisms to fight these pathogenic organisms. One important line of defense that plants have to fight these pathogens is through various inhibitors that act against these proteolytic enzymes. These inhibitors are thus active in endogenous as well as exogenous defense systems. Protease inhibitors active against different mechanistic classes of proteases have been classified into different families on the basis of significant sequence similarities and structural relationships. Specific protease inhibitors are currently being overexpressed in certain transgenic plants to protect them against invaders. The current knowledge about plant protease inhibitors, their structure and their role in plant defense is briefly reviewed.
... The peptides differ in their P 1 residue, which is known to be a major determinant of inhibitory speci®city (15,16). ...
Four 11-residue peptides based on the Bowman–Birk inhibitor (BBI) structure were synthesized. These were tested for their ability to inhibit human β-tryptase. Peptides with a basic residue at P1 inhibited tryptase even though the intact BBI protein is inactive. This result is interpreted in terms of the unique structural arrangement of active sites in tryptase which prevent access by large protein inhibitors.
... The first reactive site in these inhibitors is usually specific for trypsin, chymotrypsin and elastase (Qi et al. 2005). The active site configuration in these inhibitors is stabilized by the presence of seven conserved disulfide bonds (Chen et al. 1992;Lin et al. 1993). On the other hand BBIs from monocotyledonous plants are of two types. ...
... Fig. (1b) The Bowman-Birk family of inhibitors is another one of the well studied families of plant proteinase inhibitors; large quantities are found in the seeds of leguminous plants [11]. Several X-ray [27][28][29] and NMR [30][31][32] structures of BBI have been determined both unbound and in complex with a cognate proteinase [33][34][35][36][37]. The overall fold of the approximately 30 residues results in two domains having similar secondary structures, each having an independent reactivesite loop [35,38]. ...
This review outlines known examples of the three-dimensional structures of protein proteinase inhibitors from plants. Three families of enzymes, serine proteinases, carboxypeptidases and cysteine proteinases, are targeted by at least a dozen inhibitor families, with the majority of them adopting the standard mechanism of inhibition towards the serine proteinases. All of the inhibitors discussed maintain compact and stable inhibitory domains that bind to the active site of their target proteinases and prevent access to the substrate molecules. One interesting highlight is the knottin group. Three separate inhibitor families utilize the overall knottin fold in a different way. This fold can accommodate extensive sequence variation and for each of the squash, Mirabilis and Potato carboxypeptidase families, the proteinase-binding residues are found at a different location. Plants have also evolved additional strategies to regulate proteinase activity, such as linking inhibitory domains and targeting multiple enzymes at once. The structural aspects of these strategies are discussed in the review.
... Interactions of proteases and the 181 amino acid SBTI are well understood on functional and structural level, not least because its reactive P1 Arg- P1 0 Ile bond was an early target of bioengineering [250,251]. The much shorter LBTI comprises 71 residues with a P1 Lys-P1 0 Ser reactive bond for trypsin-like and a P1 Leu-P1 0 Ser for chymotrypsin-like proteases, but some related Bowman-Birk inhibitors possess a second reactive bond with P1 Arg [252,253]. Due to its anticarcinogenic effects this inhibitor type from plant nutrients gained much interest in medical sciences [254]. SBTI efficiently inhibited the tryptic KLKs 5 and 14 at an IC 50 of 100 nM, while KLK6 required only 16 nM for 82% inhibition and KLK4 about 50 mM for 99% inhibition [15,48,174]. ...
Including the true tissue kallikrein KLK1, kallikrein-related peptidases (KLKs) represent a family of fifteen mammalian serine proteases. While the physiological roles of several KLKs have been at least partially elucidated, their activation and regulation remain largely unclear. This obscurity may be related to the fact that a given KLK fulfills many different tasks in diverse fetal and adult tissues, and consequently, the timescale of some of their physiological actions varies significantly. To date, a variety of endogenous inhibitors that target distinct KLKs have been identified. Among them are the attenuating Zn(2+) ions, active site-directed proteinaceous inhibitors, such as serpins and the Kazal-type inhibitors, or the huge, unspecific compartment forming α(2)-macroglobulin. Failure of these inhibitory systems can lead to certain pathophysiological conditions. One of the most prominent examples is the Netherton syndrome, which is caused by dysfunctional domains of the Kazal-type inhibitor LEKTI-1 which fail to appropriately regulate KLKs in the skin. Small synthetic inhibitory compounds and natural polypeptidic exogenous inhibitors have been widely employed to characterize the activity and substrate specificity of KLKs and to further investigate their structures and biophysical properties. Overall, this knowledge leads not only to a better understanding of the physiological tasks of KLKs, but is also a strong fundament for the synthesis of small compound drugs and engineered biomolecules for pharmaceutical approaches. In several types of cancer, KLKs have been found to be overexpressed, which makes them clinically relevant biomarkers for prognosis and monitoring. Thus, down regulation of excessive KLK activity in cancer and in skin diseases by small inhibitor compounds may represent attractive therapeutical approaches.
... The deduced MBTI sequence was compared with the previously determined sequence [24,27] and the MBTI-F reported by Wilson et al. [28] (PRF accession number 0907248A) (Fig. 3). The results showed that the deduced sequence was basically consistent with the determined sequence, except for six undetermined residues at the N-terminus and two additional Asp residues at the C-terminus. ...
The Bowman-Birk protease inhibitors have recently attracted attention for their potential as cancer preventive and suppressing agents. They contain two canonical binding loops, both consisting of nine highly conserved residues capable of inhibiting corresponding serine proteases. In this study, we cloned the cDNA of the mung bean trypsin inhibitor, one of the most studied Bowman-Birk protease inhibitors. A modified peptide, Lys33GP, with 33 residues derived from the long chain of the Lys active fragment of mung bean trypsin inhibitor, was successfully expressed in Escherichia coli as a glutathione-S-transferase fusion protein. The recombinant product was obtained with a high yield, and exhibited potent inhibitory activity. Meanwhile, a shorter peptide composed of only 16 residues (the Lys16 peptide), corresponding to the active core of the fragment, was synthesized. Both the recombinant and the synthesized peptides had the same inhibitory activity toward trypsin at a molar ratio of 1 : 1, implying that the Lys16 peptide with two disulfide bonds is possibly the essential structural unit for inhibitory activity. Using site-directed mutagenesis, the P(1) position Lys was replaced by Phe, and the resulting mutant, Lys33K/F, was determined to have potent chymotrypsin inhibitory activity. Both Lys33GP and the Lys33K/F mutant may be potential pharmaceutical agents for the prevention of oncogenesis.
Dengue fever is an acute viral infection caused by the mosquito of the Aedes genus, with the largest incidence in tropical and sub-tropical regions of the world. There are no specific treatments against the dengue virus, and only the symptoms resulting from the disease are treated. The NS3/NS2B serine protease enzyme is essential for the life cycle of the virus and a promising target for drug development against the virus. In this work, acrylamide derivatives obtained from the literature data were submitted to molecular modeling studies. According to the results of the molecular docking calculations and subsequent consensual analysis, which consists of a statistical treatment used to assign the same importance to the results from different programs, and visual inspection we selected three compounds, which make hydrogen bonds with the amino acid residues His51, Asp75, Ser135, Gly151 and Gly153. Thus, Molecular Dynamics simulations were performed with three selected ligands using the hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) method, which the semiempirical AM1 Hamiltonian was used to describe the QM part and the OPLS-AA force field as method of Molecular Mechanics . The Root Mean Square Deviation (RMSD) graphs showed that systems were stabilized in 10 ns. Also, through the contribution of individual residues we observed that key residues, such as Ser34, Trp50, Lys73 and Gly151, were responsible for stabilization of the complex in the active site of the dengue enzyme.
Serine protease inhibitors are extremely widespread and are found in bacteria as well as in eukaryotic cells. They are divided into at least 13 distinct classes (for reviews, see Laskowski and Kato, 1980; Laskowski, 1986; Laskowski et al., 1987a–b; Richardson, 1991). Representative three-dimensional structures have been determined for most of these families (Bode and Huber, 1992). Although their overall protein folds are different, their common feature is a structurally conserved binding loop, which binds in the active site of the corresponding serine protease in a substrate-like fashion. Three-dimensional structures of two serine protease inhibitors are described in this chapter: LDTI, leech derived tryptase inhibitor and RBI, the bifunctional α-amylase/trypsin inhibitor from seeds of ragi. The characteristic structural properties of these inhibitors are illuminated in terms of their interaction with target proteases.
The complexity of the physical interactions which govern macromolecules, along with natural selection processes, has driven the evolution of the subtly balanced biochemical systems seen in biology, and as part of this, protein behaviour ranging from surprising to mysterious. The balances of the complex enzymatic mixtures found in vivo enable sensitive and rapid triggering of biochemical response mechanisms, for example, while maintaining the stability and adaptability required for life. This combination of stability and subtlety is found among protein structures as well: a few amino acid types with unlimited variety in sequences; a few types of secondary structure but many globular folds; common folding scaffolds (predictable?) with hypervariability at functional segments.
The major Bowman-Birk inhibitor (BBIs) of horsegram (Dolichos biflorus) HGI-III, contains seven interweaving disulfides is extremely stable to high temperatures. The contributions of two disulfide bonds in the trypsin domain to thermal stability and functionality were evaluated using disulfide deletion variants wild type protein. Thermal denaturation kinetics, differential scanning calorimetry and urea denaturation studies indicate that the absence of either of the two disulfides destabilizes the protein significantly. C20-C66 contributes substantially to both thermal stability and controls trypsin and chymotrypsin inhibitor activity. These two disulfides act in synergy as deletion of both disulfides leads to a complete loss of thermal stability. The data indicate that the two subdomains are not entirely independent of each other. Long range interactions, between the domains are facilitated by C20-C66. The deletion of the disulfide bonds also increased proteolytic susceptibility in a manner similar to the decreased thermal stability. From this study of rHGI a prototype of legume BBIs in can be concluded that among the array of seven evolutionarily conserved disulfide bonds, the disulfide C20-C66 that connects a residue in the trypsin domain with a residue at the border of the same domain plays a dominant role in maintaining functional and structural stability.
The role of the P2′ residue in proteinase inhibitors of the Bowman-Birk family was investigated using synthetic cyclic peptides based on the reactive site loop of the inhibitor. A series of 21 variants having different P2′ residues was tested for inhibition of trypsin, and the rate at which they were hydrolysed by this enzyme was also measured. Variation at P2′ was found to result in marked differences in inhibitory potency, with the best sequence (Ile) having a Ki value of 9 nM. Peptides with P2′ Gly, Pro or Glu failed to demonstrate any measurable inhibition (Ki>1 mM). The peptides also displayed significant differences in the rates at which they were hydrolysed, which varied by over three orders of magnitude between the difference sequences. There was found to be overall correlation between the Ki value and the rate of hydrolysis, with peptides that inhibited best also being hydrolysed more slowly. The results are discussed in light of the sequence information for Bowman-Birk inhibitor proteins.
Nine species of Leguminosae seeds from Amazon forest have been screened for trypsin and chymotrypsin inhibitory activity. The species involved were Cassia basselari, Cassia grandis, Cassia occidentalis, Dialium guianense, Inga fagifolia, Inga rubiginosa, Inga umbratica, Inga velutina and Mimosa guillandinae. Extracts from all the species, except I. umbratica, contained significant activity against trypsin, chymotrypsin and blood human clotting factors Xa. Two new chymotrypsin inhibitors (IuCI-1 and IuCI-2) have been purified to homogeneity from I. umbratica with molecular masses of 20,088.6 and 20,271.2, respectively.
The nematodes like root-knot and cyst are plant-parasitic pest found in horticultural and agricultural crops. They do damages in the roots of plants as a result losses million tons of production. High cost of nematicides and environment safety concern has necessitated finding of some alternative methods. Under Integrated Pest Management (IPM) such problems are solving significantly by means of target gene inhibition, agrobacterium mediated transformation etc. One of this strategy use Plant Proteinase Inhibitors (PIs) gene which are used to control the proteolysis mechanism of Pest by inhibiting gut Serine Proteinase (SP). Present work investigates the utility of computer aided methods to study the mechanism of Protein-Protein interactions and thereby inhibition of Serine Proteinase by PIs. Hence 3D models of Serine Proteinase as well as Serine Proteinase Inhibitors (SPIs) generated using homology modeling. Validations of constructed models have been done by PROCHECK, VERIFY3D, ERRAT and PROSA. Prediction of Protein interacting surface patches and site specific protein docking was performed by using ZDOCK Server. Backbone refinement of output protein complexes was executed in Fiber Dock server. Interaction study between SP and SPIs complexes shows their comparative inhibition efficacy, measured in terms of number of hydrogen bonds, Van dar wall attraction and docking energy. This work reported that Vigna marina and Phaseolus oligospermus are having better inhibition efficiency in comparison to other inhibitors.
Four proteinase inhibitors were purified to homogeneity from horse gram (Dolichos biflorus). These inhibitors are double-headed and inhibit trypsin and chymotrypsin simultaneously and independently. Dissociation constants range between 0.87 and 4.6 × 10−7 M. Each of the four isoinhibitors possesses a crucial lysine residue at the trypsin reactive site. These inhibitors have molecular masses of 8.5 kDa and isoelectric points of 4.6 to 5.6. They exist mainly as dimers under physiological conditions. Amino acid analysis revealed high levels of half-cystine, serine, aspartate and proline but low levels of methionine and aromatic amino acids. Amino-terminal sequence analysis revealed that each of the four isoinhibitors have a conserved core sequence but are divergent at the N-terminal end. These inhibitors belong to the Bowman-Birk (BBI) family of proteinase inhibitors as reflected by their inhibitory properties, amino acid composition and homology to other BBIs.
The principal objective of this work was to distinguish between kinetic and thermodynamic reaction control in protein folding. The deleterious effects of a specific mutation on spontaneous refolding competence were analyzed for this purpose. A Bowman-Birk-type proteinase inhibitor of trypsin and chymotrypsin was selected as a double-headed model protein to facilitate the detection of functional irregularities by the use of functional assays. The parent protein spontaneously folds into a single, fully active and thermodynamically stable state in a redox buffer after reduction/denaturation. By contrast, the properties of a P′1Ser→Pro variant in the trypsin-reactive subdomain differ before and after refolding on trypsin-Sepharose. A heterogenous and thermodynamically dominant population of conformers was attained in solution. However, the enzyme-inhibitory activity of the variant was dramatically increased in the presence of trypsin-Sepharose and a stoichiometric ratio of the two subdomains was obtained as expected for a single conformation. The subsequent return for the initial mixture of conformers in solution reveals a high kinetic barrier late in the folding process. The template facilitates folding kinetically, as shown by a rate acceleration of more than four orders of magnitude. The final state was also the thermodynamically favoured one on the template, due to its increased affinity for the enzyme. The long-range effects on folding kinetics and the partial activity, and the absence of free sulfhydryl groups after refolding in solution indicate rearrangements between closely related conformers late in folding. The importance of minor structural distortions in immobilized trypsin suggests a close structural analogy between the final and the transition state of protein folding.
A peptide mixture containing 21 peptide sequences has been constructed to test the Bowman-Birk inhibitor reactive-site loop motif as the basis of inhibition for a range of serine proteases. The 21 peptides are all based on an 11 amino acid sequence designed from a Bowman-Birk like inhibitor reactive-site loop. Variation has been introduced at the P1 site of the loop, which has been randomised to include all the natural l-amino acids (except for cysteine), plus the non-natural l-amino acids ornithine and norleucine. The mixture of peptides was screened for specific binding to immobilised porcine pancreatic elastase, subtilisin BPN′, -chymotrypsin, trypsin, anhydro--chymotrypsin and anhydrotrypsin. Five peptides from the mixture bind to -chymotrypsin, two of which also bind to anhydro--chymotrypsin, and two peptides bind trypsin, neither of which binds to anhydro-trypsin. The competitive inhibition constants (K1) and the rates of proteolytic hydrolysis of the individual peptides with their respective enzymes were determined. The rates of hydrolysis were found to vary widely and show little correlation with the K1 values. In the case of the -chymotrypsin inhibitors, the peptides with the lowest Ki (0.1–0.05 mM) were the only peptides that bound to anhydro--chymotrypsin. However, no peptides bound to anhydrotrypsin, suggesting a fundamental difference in the way that -chymotrypsin and trypsin are inhibited by these cyclic peptides.
The Bowman-Birk family of proteinase inhibitors from seeds of leguminous plants usually have a molecular mass of 8000 to 10,000 Da. Horse gram (Dolichos bifloros or Macrotyloma uniflorum) seeds contain an unusual Bowman-Birk inhibitor of molecular mass 15,500 Da active against both trypsin and chymotrypsin. In order to elucidate its three-dimensional structure, its evolutionary relationship with the more usual Bowman-Birk inhibitors and to study the structure-function properties, this inhibitor has been purified and crystallized. The purified protein crystallizes easily under a variety of conditions in different crystal forms. Crystals obtained by precipitating the protein (3 to 5 mg/ml in 50mM Tris·HCl (pH 8·0)) with 5% ammonium sulphate and 2 to 3% PEG 4000 appear to be suitable for structure determination by X-ray diffraction. The crystals belong to cubic space group P213 (a=110·81 Å) and diffract X-rays to beyond 3·0 Å resolution.
The Bowman-Birk trypsin inhibitor from barley seeds (BBBI) consists of 125 amino acid residues with two inhibitory loops. Its crystal structure in the free state has been determined by the multiwavelength anomalous diffraction (MAD) method and has been refined to a crystallographic R-value of 19.1 % for 8.0–1.9 Å data. This is the first report on the structure of a 16 kDa double-headed Bowman-Birk inhibitor (BBI) from monocotyledonous plants and provides the highest resolution picture of a BBI to date. The BBBI structure consists of 11 β-strands and the loops connecting these β-strands but it lacks α-helices. BBBI folds into two compact domains of similar tertiary structure. Each domain shares the same overall fold with 8 kDa dicotyledonous BBIs. The five disulfide bridges in each domain are a subset of the seven disulfide bridges in 8 kDa dicotyledonous BBIs. Two buried water molecules form hydrogen bonds to backbone atoms in the core of each domain. One interesting feature of this two-domain inhibitor structure is that the two P1 residues (Arg17 and Arg76) are approximately 40 Å apart, allowing the two reactive-site loops to bind to and to inhibit two trypsin molecules simultaneously and independently. The conformations of the reactive-site loops of BBBI are highly similar to those of other substrate-like inhibitors. This structure provides the framework for modeling of the 1:2 complex between BBBI and trypsin.
A series of hK6 inhibitors with a para-amidobenzylamine P1 group and a 2-hydroxybenzamide scaffold linker was discovered through virtual screening. The X-ray structure of hK6 complexed with compound 9b was determined to a resolution of 1.68Å. The tertiary folding of the hK6 complexed with the inhibitor is conserved relative to the structure of the apo-protein, whereas the interaction between hK6 and the inhibitor is consistent with both the SAR and the in silico model used in the virtual screening.
The Bowman-Birk inhibitors (BBIs) are currently investigated with renewed interest due to their therapeutic properties in cancer and other inflammatory disease treatment. The molecular mass of the BBI is a limitation, as sufficient amounts of the inhibitor do not reach the organs outside the gastrointestinal tract when administered orally.
The anti-tryptic domain of HGI-III of horsegram (Dolichos biflorus) was cloned using the vector pET-20b (+) and expressed in E. coli BL21 (DE3) pLysS.
Kinetic analysis of this anti-tryptic peptide (recombinant trypsin inhibitory domain (rTID)) reveals that it is a potent inhibitor of trypsin and human tryptase. The K(i) (3.2 ± 0.17 × 10(-8) M) establishes a very high affinity to bovine trypsin. rTID inhibited human lung tryptase (IC(50) 3.78 ± 0.23 × 10(-7) M). The rTID is resistant to the digestive enzymes found in humans and animals.
These properties propagate further research on the use of rTID as a therapeutic for cancer and other related inflammatory diseases.
The unique properties of silk fibroin were combined with keratin to develop new wound-dressing materials. Silk fibroin/keratin (SF/K) films were prepared to reduce high levels of elastase found on chronic wounds. This improved biological function was achieved by the incorporation of a small peptide synthesized based on the reactive-site loop of the Bowman-Birk Inhibitor (BBI) protein. In vitro degradation and release were evaluated using porcine pancreatic elastase (PPE) solution as a model of wound exudate. It was found that biological degradation and release rate are highly dependent on film composition. Furthermore, the level of PPE activity can be tuned by changing the film composition, thus showing an innovative way of controlling the elastase-antielastase imbalance found on chronic wounds.
Pest insects such as Helicoverpa spp. frequently feed on plants expressing protease inhibitors. Apparently, their digestive system can adapt to the presence of protease inhibitors. To study this, a trypsin enzyme was purified from the gut of insects that were raised on an inhibitor-containing diet. The amino-acid sequence of this enzyme was analysed by tandem MS, which allowed assignment of 66% of the mature protein amino acid sequence. This trypsin, called HzTrypsin-S, corresponded to a known cDNA sequence from Helicoverpa. The amino acid sequence is closely related (76% identical) to that of a trypsin, HzTrypsin-C, which was purified and identified in a similar way from insects raised on a diet without additional inhibitor. The digestive properties of HzTrypsin-S and HzTrypsin-C were compared. Both trypsins appeared to be equally efficient in degrading protein. Four typical plant inhibitors were tested in enzymatic measurements. HzTrypsin-S could not be inhibited by > 1000-fold molar excess of any of these. The same inhibitors inhibited HzTrypsin-C with apparent equilibrium dissociation constants ranging from 1 nM to 30 nM. Thus, HzTrypsin-S seems to allow the insect to overcome different defensive proteinase inhibitors in plants.
The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively
characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a
ternary complex at 2.48 Å resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 Å apart. The
overall fold of API-A belongs to the β-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The
two P1 residues were unambiguously assigned as Leu87 and Lys145, and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met83 to P5′ Ala92, adopts a novel conformation with the Leu87 completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting
of residues P5 Cys141 to P5′ Glu150, binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds
light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering
of the reactive sites.
The principal objective of this work was to distinguish between kinetic and thermodynamic reaction control in protein folding. The deleterious effects of a specific mutation on spontaneous refolding competence were analyzed for this purpose. A Bowman-Birk-type proteinase inhibitor of trypsin and chymotrypsin was selected as a double-headed model protein to facilitate the detection of functional irregularities by the use of functional assays. The parent protein spontaneously folds into a single, fully active and thermodynamically stable state in a redox buffer after reduction/denaturation. By contrast, the properties of a P'1Ser-->Pro variant in the trypsin-reactive subdomain differ before and after refolding on trypsin-Sepharose. A heterogenous and thermodynamically dominant population of conformers was attained in solution. However, the enzyme-inhibitory activity of the variant was dramatically increased in the presence of trypsin-Sepharose and a stoichiometric ratio of the two subdomains was obtained as expected for a single conformation. The subsequent return for the initial mixture of conformers in solution reveals a high kinetic barrier late in the folding process. The template facilitates folding kinetically, as shown by a rate acceleration of more than four orders of magnitude. The final state was also the thermodynamically favoured one on the template, due to its increased affinity for the enzyme. The long-range effects on folding kinetics and the partial activity, and the absence of free sulfhydryl groups after refolding in solution indicate rearrangements between closely related conformers late in folding. The importance of minor structural distortions in immobilized trypsin suggests a close structural analogy between the final and the transition state of protein folding.
The structural model derived from X-ray crystallography for unphosphorylated wild-type chicken cystatin is compared with two chicken cystatin structures derived from NMR spectroscopy: the phosphorylated wild-type and the genetically engineered variant AEF-SIM-M29I-M89L. The comparison shows the same overall fold, but also significant differences in structurally variable segments of the polypeptide chain. The largest such segment, comprising residues 71 to 89, is a region characteristic of the family 2 cystatin inhibitors which contains a disulphide bridge (71-81) and the phosphorylation site (Ser80) discussed in the accompanying article. In the crystal structure, the segment 71 to 76 is found as a flexible loop, 77 to 85 as an alpha-helical segment, and 86 to 89 is completely undefined. The solution NMR structures on the other hand are disordered in the initial segment 72 to 80, have an extended conformation at 81 to 83 in contact with the beta-sheet, and clearly show a beta-turn at residues 87 to 90. The segment comprising residues 53 to 57, with smaller variability, is of particular interest as the hairpin loop conserved throughout the cystatin superfamily which binds to the cysteine proteinase. In most of the solution NMR structures, this segment adopts a conformation more like that of stefin B, a family 1 cystatin inhibitor, as was observed in the crystal structure of its inhibitory complex with papain. The differences between the structures are rationalized by an examination of the crystal contacts generated by hypothetical crystal packing of the NMR structures. Additionally, the X-ray refinement shows evidence of conformational disorder in the crystal. Joint refinement with NOE restraints and reflection data does not produce a structure to satisfy the restraints of both methods.
Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that of Macrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.
The three-dimensional structure of the Bowman-Birk type proteinase inhibitor (PI-II) has been determined by x-ray crystallography and refined at 2.5-A resolution. This protein is a specific inhibitor of trypsin. Two reactive site loops, one at each end of the PI-II molecule, are structurally similar to each other and to reactive-site loops of pancreatic secretory trypsin inhibitor (Bolognesi, M., Gatti, G., Menegatti, E., Guarneri, M., Marquart, M., Papamokos, E., and Huber, R. (1982) J. Mol. Biol. 162, 839-869) and bovine pancreatic trypsin inhibitor (Deisenhofer, J., and Steigemann, W. (1975) Acta Crystallogr. B31, 238-250). PI-II is the first reported Bowman-Birk type inhibitor structure to be refined at high resolution, providing further insight into inhibitor mechanisms.
The crystal structure of the complex formed by bovine trypsin and Bowman-Birk type protease inhibitor AB-I extracted from azuki beans (Vigna angularis) “Takara” has been analyzed. The structure was solved by the application of the phase combination of single isomorphous phases and trypsin model phases, followed by phase improvement using the iterative Fourier technique. From the resulting electron density map, a three-dimensional atomic model of the trypsin binding domain of AB-I has been built. The peptide chain at the trypsin reactive site turns back sharply at Pro29 and forms a 9-residue ring (Cys24–Cys32). The “front side” of this ring, consisting of the reactive site (Cys24–Met28), interacts with trypsin in a similar manner to other families of inhibitors and forms a stable complex, which seems to be maintained by the interactions with the “back side” of this ring (Pro29–Cys34). The similar spatial arrangements of the “back side” of this inhibitor and the “secondary contact region” of the other inhibitors with respect to the reactive site suggest an important common role of these regions in exhibiting inhibitory activity.
The three-dimensional structure of the Bowman-Birk type proteinase inhibitor (PI-II) has been determined by x-ray crystallography and refined at 2.5-A resolution. This protein is a specific inhibitor of trypsin. Two reactive site loops, one at each end of the PI-II molecule, are structurally similar to each other and to reactive-site loops of pancreatic secretory trypsin inhibitor (Bolognesi, M., Gatti, G., Menegatti, E., Guarneri, M., Marquart, M., Papamokos, E., and Huber, R. (1982) J. Mol. Biol. 162, 839-869) and bovine pancreatic trypsin inhibitor (Deisenhofer, J., and Steigemann, W. (1975) Acta Crystallogr. B31, 238-250). PI-II is the first reported Bowman-Birk type inhibitor structure to be refined at high resolution, providing further insight into inhibitor mechanisms.
Bond-length and bond-angle parameters are derived from a statistical survey of X-ray structures of small compounds from the Cambridge Structural Database. The side chains of the common amino acids and the polypeptide backbone were represented by appropriate chemical fragments taken from the Database. Average bond lengths and bond angles are determined from the resulting samples and the sample standard deviations provide information regarding the expected variability of the average values which can be parametrized as force constants. These parameters are ideally suited for the refinement of protein structures determined by X-ray crystallography since they are derived from X-ray structures, are accurate to within the deviations from target values suggested for X-ray structure refinement and use force constants which directly reflect the variability or uncertainty of the average values. Tests of refinement of the structures of BPTI and phycocyanin demonstrate the integrity of the parameters and comparisons of equivalent refinements with XPLOR parameters show improvement in R factors and geometry statistics.
A model building and refinement system is described for use with a Vector General 3400 display. The system allows the user to build models using guide atoms and angles to arrive at the final conformation. It has been used to assist in difference Fourier map interpretation at medium and high resolution, and to build a protein molecule into a multiple isomorphous replacement phased electron density map.
A rigid-body refinement method and program for crystallography of macromolecules is described. Orientational and translational parameters are refined by fitting the molecular Fourier transforms to the observed structure-factor amplitudes. The range of convergence of the method has been tested on four examples with known crystal structure: PTI, chymotrypsinogen and two forms of α1 anti-trypsin. It was successfully applied in the structure solution of two unknown crystal structures: a third form of α1 anti-trypsin and C-phycocyanin.
The trypsin-reactive subdomain of the Bowman-Birk-inhibitor of serine proteinases is shown to be highly vulnerable to perturbations induced by single amino acid replacements. Dramatic deviations from a stoichiometric 1:1 ratio to the chymotrypsin-reactive subdomain used as an internal standard occur with all variants after renaturation in solution. A stoichiometric 1:1 ratio of subdomains is achieved via a new refolding procedure on trypsin-Sepharose as a matrix with complementary structure.
The crystal structure of benzamidine-inhibited bovine β-trypsin has been refined by constrained crystallographic refinement at 1·8 Å resolution. The final R-value is 0·23. The estimated standard deviation of the atomic co-ordinates is less than 0·1 Å. A preliminary refinement of inhibitor-free trypsin at 1·5 Å resolution shows only very slight conformational changes to benzamidine-inhibited β-trypsin.The differences between internal main-chain atoms of free trypsin and the trypsin component in the trypsin-inhibitor complex (Huber et al., 1974a) is 0·26 Å, <Δø}, <Δψ> are 8·0°. Complex formation does not significantly influence the peptide chain folding and only slightly alters the active site residues. In particular, Ser 195 Oγ does not change its dihedral angle considerably.The refinement led to the finding of a single site occupied by calcium (Bode & Schwager, 1975). The peptide chain folding, hydrogen bond pattern and the location of internal solvent is very similar to free α-chymotrypsin (Birktoft & Blow, 1972). Ser 195 Oγ, however, differs by about 180° in dihedral angle.A comparison with the partial atomic co-ordinate list given by Stroud et al. (1974) and Krieger et al. (1974) for benzamidine-inhibited bovine trypsin shows large discrepancies in the specificity site Asp189, Ser190, Gln192 and the active site-associated solvent.
Five protease inhibitors, I–V, in the molecular weight range 7000–8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I–V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibits chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residues of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.
Four Bowman-Birk type double-headed inhibitors (B, C-II, D-II, and E-I) were isolated from soybeans. Inhibitor B was different from Bowman-Birk inhibitor only in chromatographic behavior. One mole of C-II inhibited one mole each of bovine trypsin and bovine alpha-chymotrypsin, probably at the same site, and porcine elastase at another reactive site. In the ordinary assay system D-II and E-I inhibited only trypsin activity at a non-stoichiometric inhibitor-enzyme ratio of 1:1.4, and the complexes had rather high dissociation constants. These inhibitors were all inactive toward subtilisin BPN'.
Soybean inhibitor D-II is an inhibitor of bovine trypsin. Sequence analysis was carried out on the reduced and S-carboxymethylated protein by conventional methods to establish the complete amino acid sequence. The sequence of D-II indicated high homology with other legume inhibitors, but it was unique because of the occurrence of identical residues (arginine) at both of the reactive sites. This structure is thought to reflect that of a prototype double headed inhibitor. The possible evolutionary process of the legume double-headed inhibitors is discussed on this basis.
Comparison with another soybean inhibitor C-II suggested that a single methionine (C-II)—glutamine (D-II) replacement at the P2′ position resulted in the loss of α-chymotrypsin inhibitory activity of D-II. The results of a hydrogen peroxide oxidation experiment on C-II supported this suggestion.
The sequence of the amino-terminal 21 residues of inhibitor E-I was determined using a sequenator. It was shown that this inhibitor lacks the amino-terminal nine residues of D-II.
The substrate-like 'canonical' inhibition by the 'small' serine proteinase inhibitors and the product-like inhibition by the carboxypeptidase inhibitor have provided the only atomic models of protein inhibitor--proteinase interactions for about 15 years. The recently published structures of cystatin/stefin--papain complexes and of hirudin--thrombin complexes reveal novel non-substrate-like interactions. In addition, the structure of pro-carboxypeptidase shows a model of inactivation which bears resemblance to proteinase/protein inhibitor systems. Considerable progress in understanding the transition between native and cleaved states of the serpins has also been made by several recent structural studies.
The three-dimensional structure of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in solution has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing using the program XPLOR. The structure was defined by 907 NOEs involving intra- and interresidue contacts which served as distance constraints for a protocol of dynamical simulated annealing. In addition, 48 phi angle constraints involving non-proline amino acids, 29 chi angle constraints, six omega angle constraints for the X-Pro peptide bond, and 35 stereoassignments for prochiral centers were incorporated during the course of the calculation. The protein is characterized by two distinct binding domains for serine protease. Each domain is comprised of a beta-hairpin (antiparallel beta-sheet and a cis-proline-containing type VIb reverse turn) with a short segment making a third strand of antiparallel beta-sheet. The structure determination and refinement are described, and the structure is compared to other structures of Bowman-Birk inhibitors as well as other families of serine protease inhibitors.
Conformational disorder in crystal structures of ribonuclease-A and crambin is studied by including two independent structures in least-squares optimizations against X-ray data. The optimizations are carried out by X-ray restrained molecular dynamics (simulated annealing refinement) and by conventional least-squares optimization. Starting from two identical structures, the optimizations against X-ray data lead to significant deviations between the two, with rms backbone displacements of 0.45 A for refinement of ribonuclease at 1.53 A resolution, and 0.31 A for crambin at 0.945 A. More than 15 independent X-ray restrained molecular dynamics runs have been carried out for ribonuclease, and the displacements between the resulting structures are highly reproducible for most atoms. These include residues with two or more conformations with significant dihedral angle differences and alternative hydrogen bonding, as well as groups of residues that undergo displacements that are suggestive of rigid-body librations. The crystallographic R-values obtained are approximately 13%, as compared to 15.3% for a comparable refinement with a single structure. Least-squares optimization without an intervening restrained molecular dynamics stage is sufficient to reproduce most of the observed displacements. Similar results are obtained for crambin, where the higher resolution of the X-ray data allows for refinement of unconstrained individual anisotropic temperature factors. These are shown to be correlated with the displacements in the two-structure refinements.
Crystallographic refinement by simulated annealing with molecular dynamics has been applied to a 2.8 A (1 A = 0.1 nm) resolution X-ray structure of aspartate aminotransferase. Comparison of the refined structure and a structure obtained by combined restrained least-squares refinement and manual re-fitting shows a similar R factor, stereochemistry, and mean difference from the isomorphous replacement phase centroids. Crystallographic refinement by simulated annealing accomplished structural changes and improvements of the electron density maps that were not possible by using restrained least-squares refinement without manual re-fitting. Crystallographic refinement by simulated annealing can generate an ensemble of structures, each of which agrees with the diffraction information. Regions of large variations of the ensemble indicate either erroneously fitted or disordered segments of the macromolecule.
The structure of Bowman-Birk type protease inhibitor (A-II from peanut) is described at 3.3 A resolution. The molecules form a tetramer with 222 local symmetry in our crystals. Each monomer has an elongated shape with approximate dimensions of 45 X 15 X 15 A and consists of two distinct domains. The three-dimensional structures of the two domains are similar and are related by the intramolecular approximate twofold rotation axis. The two independent protease binding sites protrude from the molecular body on opposite sides. A scheme for the molecular evolution of the double-headed Bowman-Birk type protease inhibitors is proposed, based on the three-dimensional structure.
The biosynthesis of three major peroxisomal membrane polypeptides of rat liver was investigated. Total hepatic RNA extracted by the guanidinium/CsCl method from three control and three di(2-ethylhexyl)phthalate (a peroxisomal proliferator)-fed rats was translated in vitro in a rabbit reticulocyte lysate protein-synthesizing system. Translation products were immunoprecipitated by the antibodies against peroxisomal membrane polypeptides, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and analyzed by fluorography. The in vitro translation products of 70, 26, and 22 kDa peroxisomal membrane polypeptides were apparently of the same size as the respective mature polypeptides. The ratio of translatable mRNA levels for the 70, 26, and 22 kDa polypeptides in di(2-ethylhexyl)phthalate-fed rats to those in control rats were 5.4, 11.4, and 2.7, respectively. The synthesis of these three polypeptides with the free polysome fraction from di(2-ethylhexyl)phthalate-fed rats was more active than that with the membrane-bound polysome fraction, whereas the synthesis of albumin with the free polysome fraction was 27% of that with the membrane-bound polysome fraction. These results indicate that the peroxisomal major membrane polypeptides are synthesized on free polysomes and transported to peroxisomal membrane without any apparent proteolytic processing, and that the induction of these polypeptides by administration of a peroxisomal proliferator corresponds well to the induction of the peroxisomal beta-oxidation enzymes. The data also support the idea that peroxisomes are organized from pre-existing peroxisomes.
Four decades of studies on the isolation, characterization, properties, structure, function and possible uses of the Bowman-Birk trypsin- and chymotrypsin-inhibitor from soybeans are reviewed. Starting from Bowman's Acetone Insoluble factor, designated Ai, AA and SBTIAA, the Bowman-Birk inhibitor (BBI) was found to be a protein molecule consisting of a chain of 71 amino acids cross linked by 7 disulfide bonds, with a tendency to self-associate. BBI possesses two independent sites of inhibition, one at Lys 16-Ser 17 against trypsin and the other at Leu 43-Ser 44 against chymotrypsin. It forms a 1:1 complex with either trypsin or chymotrypsin and a ternary complex with both enzymes. Ingestion of BBI by rats, chicks or quails affects the size and protein biosynthesis of the pancreas. Establishment of the full covalent structure of BBI revealed a high homology in the sequences around the two inhibitory sites, suggesting evolutionary gene duplication from a single-headed ancestral inhibitor. Scission of BBI by CNBr followed by pepsin results in two active fragments, one that inhibits trypsin and the other, chymotrypsin. Replacements and substitutions in the reactive sites result in changes in inhibitory activity and in specificity of inhibition. Conformation studies, labeling of BBI with a photoreactive reagent, chemical synthesis of cyclic peptides that include inhibitory sites, in vitro synthesis of BBI, and species specificity regarding the inhibited enzymes are described. The significance of BBI as a prototype of a family of inhibitors present in all legume seeds is discussed.
The complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) was crystallized from 17% ethanol at pH 7. The crystals have P212121 symmetry with a = 59.0, b = 62.2, and c = 150.5 Å. They contain one molecule of the 1:1 complex in the crystallographic asymmetric unit. Three-dimensional X-ray diffraction data were measured for these crystals and for crystals soaked with 0.3 mM K2PtCl4 1 mM K3UO2F5, 5 mM mercury salicylate, 0.2 mM (NH4)3IrCl6, and 5 mM mercury acetate. All strong reflections to 2.6 Å were measured, including almost all reflections to 3.8 A and 56% of the reflections between 3.0 and 2.6 Å. Beyond 3.8 Å only the K3UO2F5 and salicylate derivatives were useful. In the resulting electron density maps the trypsin molecule was easily identifiable, and although some parts of the inhibitor were not clear, a precise interpretation of residues 1′-93′ was made, including the contact region. Coordinates for this part of the inhibitor, and for adjacent parts of the trypsin molecule, were refined by the real-space refinement technique. Interactions made by soybean trypsin inhibitor with trypsin at P3, P2, P1, P1′, and P2′ are extremely similar to interactions observed in a crystallographic study of the complex of pancreatic trypsin inhibitor (Kunitz) with bovine trypsin (Rühlmann, A, Kukla, D., Schwager, P., Bartels, K., and Huber, R. (1973), J. Mol. Biol. 77, 417) and to the interactions which a normal polypeptide substrate is believed to make. These interactions account for two-thirds of about 300 interatomic contacts and for 13 of the 18 probable hydrogen bonds between the inhibitor and the enzyme. Interatomic distances at the active site show the complex to be in the form of a tetrahedral adduct of the scissile bond to the active serine. The strong binding energy of the inhibitor and the stabilization of the tetrahedral form result from the nature of the active site of the enzyme, which is designed to stabilize the transition state of peptide hydrolysis.
The crystal structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor has been refined with data to 1.9 Å resolution, using a procedure described by Deisenhofer & Steigemann (1974) in their refinement of the crystal structure of the free inhibitor. This procedure involves cycles consisting of phase calculation using the current atomic model, Fourier synthesis using these phases and the observed structure factor amplitudes and Diamond's real-space refinement (Diamond, 1971,1974). At various stages, difference Fourier syntheses are calculated to detect and correct gross errors in the model and to localize solvent molecules.The refinement progressed smoothly, starting with the model obtained from the isomorphous Fourier map at 2.6 Å resolution. The R-factor is 0.23 for 20,500 significantly measured reflections to 1.9 Å resolution, using an over-all temperature factor of 20 Å2. The estimated standard deviation of atomic positions is 0.09 Å.An objective assessment of the upper limit of the error in the atomic coordinates of the final model is possible by comparing the inhibitor component in the model of the complex with the refined structure of the free inhibitor (Deisenhofer & Steigemann, 1974). The mean deviation of main-chain atoms of the two molecular models in internal segments is 0.25 Å, of main-chain dihedral angles 5.1 ° and side-chain dihedral angles 6.5 °.A comparison of the trypsin component with α-chymotrypsin (Birktoft & Blow, 1972) showed a mean deviation of main-chain atoms of 0.75 Å. The structures are closely similar and the various deletions and insertions cause local structural differences only.
The structure of the complex of bovine trypsin and bovine pancreatic trypsin inhibitor has been determined by crystal structure analysis at 2.8 Å resolution. The structure is closely similar to the model predicted from the structures of the components. The complex is a tetrahedral adduct with a covalent bond between the carbonyl carbon of Lys-15I of the inhibitor and the γ-oxygen of Ser-195 of the enzyme. The imidazole of His-57 is hydrogen-bonded to Asp-102 and the bound seryl γ-oxygen in accord with the histidine being charged. The negatively charged carbonyl oxygen of Lys-15I forms two hydrogen bonds with the amide nitrogens of Gly-193 and Ser-195. Protonation of the leaving group N-H of Ala-16I to form an acyl-complex requires a conformational change of the imidazole of His-57. The tetrahedral adduct is further stabilized by hydrogen bonds between groups at the leaving group side and inhibitor and enzyme, which would be weakened in the acyl-enzyme. The kinetic data of inhibitor-enzyme interaction are reconciled with the structural model, and relations between enzyme-inhibitor interaction and productive enzyme-substrate interaction are proposed.
Soybean Bowman-Birk proteinase inhibitor contains 14 half-cystine residues in a relatively short polypeptide chain of 71 amino acid residues, and five half-cystine residues around the trypsin-inhibitory site are in absolutely identical locations to those around the chymotrypsin inhibitory site. To assign the locations of the disulfide bridges, the native inhibitor was digested with a mold acid proteinase, thermolysin, pronase or subtilisin to yield cystine peptides suitable for structural investigation. These were separated and purified by chromatography on a Bio-Gel P-4 column and by paper electrophoresis. A peptide containing a -Cys-Cys- sequence was further hydrolysed with 10 N sulfuric acid. Amino acid analyses and appropriate amino terminal analyses of the resulting cystine peptides or their oxidized derivatives revealed the positions of 7 disulfide bridges in the parent molecule, which indicated the presence of two homologous trypsin- and chymotrypsin-inhibitory regions, and an almost symmetrical structure of the inhibitor.
This chapter investigates the anatomy and taxonomy of protein structures. A protein is a polypeptide chain made up of amino acid residues linked together in a definite sequence. Amino acids are “handed,” and naturally occurring proteins contain only L-amino acids. A simple mnemonic for that purpose is the “corncrib.” The sequence of side chains determines all that is unique about a particular protein, including its biological function and its specific three-dimensional structure. The major possible routes to knowledge of three-dimensional protein structure are prediction from the amino acid sequence and analysis of spectroscopic measurements such as circular dichroism, laser Raman spectroscopy, and nuclear magnetic resonance. The analysis and discussion of protein structure is based on the results of three-dimensional X-ray crystallography of globular proteins. The basic elements of protein structures are discussed. The most useful level at which protein structures are to be categorized is the domain, as there are many cases of multiple-domain proteins in which each separate domain resembles other entire smaller proteins. The simplest type of stable protein structure consists of polypeptide backbone wrapped more or less uniformly around the outside of a single hydrophobic core. The outline of the taxonomy is also provided in the chapter.
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