Three-dimensional structure of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in solution.

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.     

Identification of a protein-binding surface by differential amide hydrogen-exchange measurements. Application to Bowman-Birk serine-protease inhibitor.

The binding surface of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in contact with alpha-chymotrypsin has been identified by measurement of the change in amide hydrogen-exchange rates between free and chymotrypsin-bound inhibitor. Exchange measurements were made for the enzyme-bound form of the inhibitor at pH 7.3, 25 degrees C using fast-flow affinity chromatography and direct measurement of exchange rates in the protein complex from one-dimensional and two-dimensional nuclear magnetic resonance spectra. The interface is characterized by a broad surface of contact involving residues 39 through 48 of the anti-chymotryptic domain beta-hairpin as well as residues 32, 33 and 37 in the anti-chymotryptic domain loop of the inhibitor. A number of residues in the anti-tryptic domain of the protein also have an altered exchange rate, suggesting that there are changes in the protein conformation upon binding to chymotrypsin. These changes in amide exchange behavior are discussed in light of a model of the complex based on the X-ray crystallographic structure of turkey ovomucoid inhibitor third domain bound to a alpha-chymotrypsin, and the structure of free Bowman-Birk inhibitor determined in solution by two-dimensional nuclear magnetic resonance spectroscopy. The chymotrypsin-binding loop of Bowman-Birk inhibitor in the model is remarkably similar to the binding loop conformation in crystal structures of enzyme-bound polypeptide chymotrypsin inhibitor-I from potatoes, turkey ovomucoid inhibitor third domain, and chymotrypsin inhibitor-II from barley seeds.     

Crystal structure of bovine trypsin and wheat germ trypsin inhibitor (I-2b) complex (2:1) at 2.3 a resolution

The Bowman-Birk trypsin inhibitor (BBI) from wheat germ (I-2b) consists of 123 amino acid residues with two inhibitory loops. The crystal structure of a bovine trypsin-wheat germ trypsin inhibitor (I-2b) complex (2:1) has been determined at 2.3 A resolution to a final R-factor of 0.177. A distance of 37.2 A between the contiguous contact loops allows them to bind and inhibit two trypsin molecules simultaneously and independently. Each domain shares the same overall fold with 8 kDa BBIs. The five disulfide bridges in each domain are a subset of seven disulfide bridges in the 8 kDa BBIs. I-2b consists of ten beta-strands and the loops connecting these strands but it lacks alpha-helices. The conformations of the contiguous contact loops of I-2b are in a heart-like structure. The reactive sites in both domains, Arg 17 and Lys 76, are located on the loop connecting anti-parallel beta-strands, beta 1/beta 2 and beta 6/beta 7. Strands beta 1 and beta 6 are in direct contact with trypsin molecules and form stable triple stranded beta-sheet structures via hydrogen bonds.     

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.     

Studies on soybean trypsin inhibitors. XIII. Preparation and characterization of active fragments from Bowman-Birk proteinase inhibitor.

Soybean Bowman-Birk inhibitor, a double-headed inhibitor of trypsin and alpha-chymotrypsin, was treated with cyanogen bromide and then pepsin to yield two inhibitory active fragments. Structural investigation showed that one of the fragments was derived from the trypsin inhibitory domain and the other from the chymotrypsin inhibitory domain of the inhibitor. In contrast to the unusual stability of the native inhibitor, the separated domains were less stable and could be inactivated with excess proteinases. These results suggest that the legume double-headed inhibitors acquired their unusual stability by duplicating an ancestral single-headed structure.     

Structure-function relationship of bromelain isoinhibitors from pineapple stem

Bromelain isoinhibitors from pineapple stem (BIs) are unique double-chain inhibitors and inhibit the cysteine proteinase bromelain competitively. The three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded anti-parallel beta-sheet. Unexpectedly, BIs were found to share similar folding and disulfide-bond connectivities not with the cystatin superfamily, but with Bowman-Birk trypsin/chymotrypsin inhibitor (BBI). The structural similarity between them suggests that BIs and BBI have evolved from a common ancestor and differentiated in function during the course of molecular evolution.     

Inhibitory properties and solution structure of a potent Bowman-Birk protease inhibitor from lentil (Lens culinaris, L) seeds

Bowman-Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil (Lens culinaris, L., var. Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization. LCTI is a 7448 Da double-headed trypsin/chymotrypsin inhibitor with dissociation constants equal to 0.54 nM and 7.25 nM for the two proteases, respectively. The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the kon and k*on values (1.1 microM-1.s-1 vs. 0.002 microM-1.s-1), respectively, for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman-Birk inhibitor (BBI) conformation with two antipodal beta-hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10-Asp26-Arg28 and Asp36-Asp52 residues. At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C-terminus, and a highly positive potential, surrounding the antitryptic domain. The segment 53-55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis. The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman-Birk inhibitor family.     

Preparation of photoreactive derivatives of trypsin-chymotrypsin inhibitors from soybeans and chick peas by selective modification of lysine residues

Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas, were prepared by selective modification of the epsilon-amino groups of lysine residues with 2-nitro-4(5)-azidophenylsulfenyl chlorides (2,4(5)-NAPS-C1). The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of the trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.     

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.     

Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean.

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 inhibit chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residued 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.     

Proteinase inhibition using small Bowman-Birk-type structures

Bowman-Birk inhibitors (BBIs) are cysteine-rich and highly cross-linked small proteins that function as specific pseudosubstrates for digestive proteinases. They typically display a "double-headed" structure containing an independent proteinase-binding loop that can bind and inhibit trypsin, chymotrypsin and elastase. In the present study, we used computational biology to study the structural characteristics and dynamics of the inhibition mechanism of the small BBI loop expressing a 35-amino acid polypeptide (ChyTB2 inhibitor) which has coding region for the mutated chymotrypsin-inhibitory site of the soybean BBI. We found that in the BBI-trypsin inhibition complex, the most important interactions are salt bridges and hydrogen bonds, whereas in the BBI-chymotrypsin inhibition complex, the most important interactions are hydrophobic. At the same time, ChyTB2 mutant structure maintained the individual functional domain structure and excellent binding/inhibiting capacities for trypsin and chymotrypsin at the same time. These results were confirmed by enzyme-linked immunosorbend assay experiments. The results showed that modeling combined with molecular dynamics is an efficient method to describe, predict and then obtain new proteinase inhibitors. For such study, however, it is necessary to start from the sequence and structure of the mutant interacting relatively strongly with both trypsin and chymotrypsin for designing the small BBI-type inhibitor against proteinases.     

Crystal structure of a 16 kDa double-headed Bowman-Birk trypsin inhibitor from barley seeds at 1.9 A 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 A 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 beta-strands and the loops connecting these beta-strands but it lacks alpha-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 A 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.     

The Bowman-Birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soybeans

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.     

Primary structure of Dioclea glabra trypsin inhibitor, DgTI, a Bowman-Birk inhibitor.

A novel serine proteinase inhibitor, DgTI, was purified from Dioclea glabra seeds by acetone precipitation, and ion-exchange and reverse phase chromatography. The inhibitor belongs to the Bowman-Birk family, and its primary sequence, determined by Edman degradation and mass spectrometry, of 67 amino acids is: SSGPCCDRCRCTKSEPPQCQCQDVRLNSCHSACEACVCSHSMPGLCSCLDITHFCHEPCKSSGDDED++ +. Although two reactive sites were determined by susceptibility to trypsin (Lys(13) and His(40)), the inhibitory function was assigned only to the first site. The inhibitor forms a 1:1 complex with trypsin, and Ki is 0.5 x 10(-9) M. Elastase, chymotrypsin, kallikreins, factor Xa, thrombin, and plasmin were not inhibited. By its properties, DgTI is a Bowman-Birk inhibitor with structural and inhibitory properties between the class of Bowman-Birk type I (with a fully active second reactive site), and Bowman-Birk type II (devoid of second reactive site).     

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.     

Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type

A number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr = 14,500) and II (Mr = 7,000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors II are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.     

Sequential resonance assignment and secondary structure determination of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors

The solution conformation of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors, has been investigated by nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments of the 1H NMR spectrum have been obtained by using a number of two-dimensional techniques for identifying through-bond and through-space (less than 5-A) connectivities. Elements of regular secondary structure have been identified on the basis of a qualitative interpretation of the nuclear Overhauser enhancement, coupling constant, and amide exchange data. These are two beta-sheet regions. One double-stranded antiparallel beta-sheet comprises residues 11-14 (strand 1) and 37-39 (strand 2). The other triple-stranded sheet is formed by two antiparallel strands comprising residues 45-49 (strand 4) and 53-57 (strand 5) connected by a turn (residues 50-52), and a small strand consisting of residues 20-22 (strand 3) that is parallel to strand 4.     

Purification, characterization, and cDNA cloning of a Bowman-Birk type trypsin inhibitor from Apios americana Medikus tubers

An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) M and 1.0 x 10(-6) M respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.