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Crystal Structure of the Bifunctional Soybean Bowman-Birk Inhibitor at 0.28-nm Resolution. Structural Peculiarities in a Folded Protein Conformation
Abstract
The Bowman-Birk inhibitor from soybean is a small protein that contains a binary arrangement of trypsin-reactive and chymotrypsin-reactive subdomains. In this report, the crystal structure of this anticarcinogenic protein has been determined to 0.28-nm resolution by molecular replacement from crystals grown at neutral pH. The crystal structure differs from a previously determined NMR structure [Werner, M. H. & Wemmer, D. E. (1992) Biochemistry 31, 999-1010] in the relative orientation of the two enzyme-insertion loops, in some details of the main chain trace, in the presence of favourable contacts in the trypsin-insertion loop, and in the orientation of several amino acid side chains. The proximity of Met27 and Gln48 in the X-ray structure contradicts the solution structure, in which these two side chains point away from each other. The significant effect of a Met27-->Ile replacement on the inhibitory activity of the chymotrypsin-reactive subdomain agrees with the X-ray structure. Exposed hydrophobic patches, the presence of charged amino acid residues, and the presence of water molecules in the protein interior are in contrast to standard proteins that comprise a hydrophobic core and exposed polar amino acids.
... A new salt bridge between K16 of sBBI and D189 of trypsin is formed upon proteinprotein interaction. [49,50] The essential S17 residue of sBBI is implicated in the intramolecular hydrogen bond with the Q21 residue in the free inhibitor (Scheme 1(a)). The binding of the inhibitor to trypsin results in the creation of a new hydrogen bond between the S17 residue of sBBI and S195 of trypsin (Scheme 1(b)). ...
... The binding of the inhibitor to trypsin results in the creation of a new hydrogen bond between the S17 residue of sBBI and S195 of trypsin (Scheme 1(b)). [49][50][51][52] The maximum activity of trypsin is observed in the pH range of 7-9. [53] The formation of the non-covalent complex between small size sBBI and trypsin was initially studied by ESI-MS. ...
... According to crystallographic studies, in the free sBBI molecule the S17 residue is involved in the intramolecular hydrogen bond with the Q21 residue, whereas a new hydrogen bond is likely formed between S17 (or S25 in the DII-type inhibitor) and S195 of trypsin upon interaction. [49,50] Thus, the reinforcement of the nucleophilicity of S17 (or S25) can be related to the changes in the microenvironment, especially to new hydrogen bond formation. Furthermore, it can be considered that the cooperativity effect appeared due to the presence of the neighboring SB between K16 (or R24 in the DII-type inhibitor) and D189 of trypsin. ...
The study of protein recognition sites is crucial for understanding the mechanisms of protein interaction. Mass spectrometry can be a method of choice for the investigation of the contact surface within the protein non-covalent complexes.
Probing the reactivity of essential amino acid residues of soybean Bowman-Birk inhibitor (sBBI) within the non-covalent sBBI/bovine trypsin complex was performed using covalent labeling by the BS3 cross-linker and charge tag with a quaternary ammonium group in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis.
Significant modulation of the reactivity of essential K16 and S17 residues in the sBBI molecule upon binding to trypsin was established. The studies of sBBI proteolytic peptides with the same structure but carrying different labels using metastable dissociation in LIFT mode demonstrated that fragmentation pathways were oriented by used modification (BS3 cross-linker or charge tag).
The effectiveness of the mass spectrometric approach including covalent modification for exploring protein-protein interaction sites has been demonstrated. The alteration of the reactivity of functionally important amino acid residues in the sBBI molecule is most likely related to changes in their microenvironment. It has been suggested that in the presence of charge tags fragmentation in LIFT mode proceeds through the formation of salt bridges between quaternary ammonium groups and acidic residues due to the occurrence of zwitterions (including basic and acidic residues). Despite the presence of one or several charge tags, fragmentation takes place yielding modulated bi /yj ion series depending on the positions of the tags. Copyright © 2014 John Wiley & Sons, Ltd.
... Da and a pI value of 5.14. When the hydropathy index was analysed by the ProtScale tool (Fig. 2D), it was possible to evidence some hydrophobic regions consistent with the formation of dimeric structures in solution [35,[63][64][65][66][67][68][69][70][71][72][73]. The protein was also found to be rich in Leucine, Glutamate, Isoleucine and Valine as deduced by the BioEdit software, presenting around 45% of hydrophobic residues which contributes to the hypothesis of the formation of dimeric structures in solution (Table 1). ...
... Based on the results observed by SDS-PAGE, we hypothesize that this inhibitor presents the tendency to self-association even at reducing conditions of the electrophoresis, resulting in the formation of a dimeric complex in solution. This behaviour was also observed for other serine protease inhibitors, and was attributed to the presence of a polar domain interface that is solvated with internal water molecules and hydrophobic patches [35,[63][64][65][66][67][68][69][70][71][72][73] and to the potential ionic interactions between the Lys/Arg and Asp residues, with the consequent formation of saline bridges that contribute to the stabilization of the dimeric structure, encouraging the association of the protease inhibitors [74][75][76]. Furthermore, the slight difference in the electrophoretic mobility observed for the expressed inhibitor compared to that predicted by in silico analysis, could be explained because the electrophoresis was performed on 20% polyacrylamide gels, in which the degree of compaction further affects the electrophoretic mobility, decreasing when compared to the mobility observed for other degrees of crosslinking of the polyacrylamide gel. ...
... We also assessed the magnitude of mispairing for saturating amounts of pulsed peptide versus endogenous processing of full length antigen following RNA electroporation in autologous iDCs as target cells. Finally, we aimed at strengthening V-domain pairing by the design of a novel disulfide bond into a scTCR- fragment [33] so as to eradicate traceable mispairing with any TCRα. The endogenous TCRα/β-chain deficient Jurkat-76 (J-76) leukemia T-cell line [26] was used to perform TCR mispairing studies. ...
... RCSB [41]) in order to strengthen the interaction between the Vα-and Vβ-domain so as to eradicate any association to TCRα. Disulfide bridges are characterized by well-defined structural constraints such as clustering of the torsion angle χ SS around 90° and its handedness, gauche and trans stereochemistry of χ i and χ j , respectively, and favourable Cα-distances between 4 and 6.2 Å for the interacting cysteines [33]. We tested 3 different approaches by introducing artificial disulfide bridges into scTCR gp100 a) directly between Vα and Vβ (Vα G121C / Vβ G49C according to IMGT nomenclature), b) between Vα and Cβ (Vα L46C / Cβ P82C), and c) between Vα and the C-terminal tail of the Gly/Ser-linker [17] at three different positions (Vα G49C / Linker G16-18C) to compensate for lack of information about the folding characteristics of the 19 aa linker (Figure 5A). ...
Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells.
... Embora intensivamente estudados sob o ponto de vista bioquímico e estrutural, o mecanismo de ação dos inibidores de serinoproteases ainda é objeto de investigação científica. Estruturas tridimensionais de outros inibidores pertencentes à família Bowman-Birk em forma livre (Werner e Wemmer, 1992;Suzuki et. al., 1993;Voss et. al., 1996;Catalano et. al., 2003) e em complexo com a tripsina (Park et. al., 2004, Barbosa et. al., 2007 Touil et. al., 2008), em atuações no sistema imune -sistema respiratório, sistema de defesa e pele, na regulação da homeostase, agentes antivirais (SARS, Hepatite C, Dengue, HIV), agentes antibactericidas (tuberculose, anthrax), agentes antifú ...
... ivo é formado por um loop conservado ligando duas fitas β. independentemente (DeLaSierra et. al., 1999;Koepke et. al., 2000;Park et. al., 2004;Song et. al., 1999). A conformação deste sítio reativo é mantida por uma rede de ligações de hidrogênio envolvendo P2 e P9, (figura 12) expondo a cadeia lateral de P1 para a interação inicial com a tripsina Voss et. al., 1996;Song et. al., 1999 ...
... and subjected for automated model- ing in SWISSMODEL (http://swissmodel.expasy.org/interactive). The PDB file generated from SWISS MODEL was used to build 3-dimensional protein model and the disulfide bridges, N-terminus, C-terminus as well as reactive loop of BBI domain for its activity ( Voss et al., 1996). ...
... The amino acid sequence of RbTI was imported into the online tool SWISS-MODEL (http://swissmodel.expasy.org/) to further analyze the tertiary structure of rice bean BBI peptide (Fig. 5A). The reactive loop was found to be conserved in RbTI BBI as reported by Voss et al. (1996). The final model included residues 45-99 (E = 1.14248e−10). ...
This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichia coli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.
... KTi is present in higher concentrations than BBi, and KTi content varies widely among different soybean genotypes, ranging from 4.28 to 15.0 mg per gram of soy flour [14,15]. Soybean KTi is a protein with a 21 kDa molecular weight that exists as a monomer, consisting of 181 amino acids linked by two disulfide cysteine residues, while BBi is a low-molecular-weight protein (7 kDa) consisting of 71 amino acids with seven disulfide cysteine residues [16,17]. The disulfide bonds in TIs increase their stability at high temperatures and decrease the availability of soybean processed foods [18]. ...
Trypsin inhibitors (TIs) in soybean seeds reduce the availability of processed soybean foods and animal feed. This study aimed to evaluate the trypsin inhibitor activity (TIA) in 999 Korean soybean accessions and conduct molecular characterization of soybean accessions with low TIA. TIA was evaluated using colorimetric analysis through a substrate–enzyme reaction. The average TIA of the 999 soybean accessions was 90.31%, ranging from 43.70% to 99.51%. Kunitz trypsin inhibitor 3 (KTi-3) gene target sequencing analysis was performed on seven soybean germplasm accessions (IT105782, IT170889, IT273590, IT274513, IT274515, IT276197, and IT022891) showing less than 60% TIA. Four soybean accessions (IT274513, IT274515, IT276197, and IT022891) exhibited the same mutations (a G-to-T transversion and an AG deletion) in the KTi-3 gene (Glyma.08g341500) in PI542044. In addition, whole-genome re-sequencing was performed on three soybean accessions with no mutations in the KTi-3 gene. Compared with the reference soybean genome, an in-frame insertion and five missense mutations were identified in the coding sequencing of the KTi-1 gene (Glyma.01g09500) in IT105782. A RT-qPCR analysis showed that the mRNA expression level of KTi-1 was reduced by approximately 17% in IT105782 during seed development. In this study, we identified a previously unreported mutation in the KTi-1 gene and developed a KASP marker using this new KTi-1 variant.
... The multimeric states of trypsin inhibitor were too identified by the atomic force microscopy analysis, which indicated that the inhibitor adopts stable and well-packed self-associated states in monomer-dimer-trimer-hexamer forms with globular-ellipsoidal shapes (Silva et al., 2005). The absence of a more hydrophobic core and the presence of the high content of disulfide bonds result in a constrained conformation that may be responsible for the remarkable stability and association exhibited by this inhibitor (da Silva et al., 2001), in covenant with other BBIs (Voss et al., 1996). It has been reported that the reactive sites that show interaction with proteases are generally located on the surface of the dimer, which most likely indicates that the dimeric form is the functional state of the molecule (Li de la Sierra et al., 1999). ...
A Bowman-Birk protease, i.e., Mucuna pruriens trypsin inhibitor (MPTI), was purified from the seeds by 55.702-fold and revealed a single trypsin inhibitor on a zymogram with a specific activity of 202.31 TIU/mg of protein. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under non-reducing conditions, the protease trypsin inhibitor fraction [i.e., trypsin inhibitor non-reducing (TINR)] exhibited molecular weights of 74 and 37 kDa, and under reducing conditions [i.e., trypsin inhibitor reducing (TIR)], 37 and 18 kDa. TINR-37 revealed protease inhibitor activity on native PAGE and 37 and 18 kDa protein bands on SDS–PAGE. TINR-74 showed peaks corresponding to 18.695, 37.39, 56.085, and 74.78 kDa on ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization/quadrupole time-of-flight-mass spectrometry (ESI/QTOF-MS). Similarly, TINR-37 displayed 18.695 and 37.39 kDa peaks. Furthermore, TIR-37 and TIR-18 exhibited peaks corresponding to 37.39 and 18.695 kDa. Multiple peaks observed by the UPLC-ESI/QTOF analysis revealed the multimeric association, confirming the characteristic and functional features of Bowman-Birk inhibitors (BBIs). The multimeric association helps to achieve more stability, thus enhancing their functional efficiency. MPTI was found to be a competitive inhibitor which again suggested that it belongs to the BBI family of inhibitors, displayed an inhibitor constant of 1.3 × 10–6 M, and further demonstrates potent anti-inflammatory activity. The study provided a comprehensive basis for the identification of multimeric associates and their therapeutic potential, which could elaborate the stability and functional efficiency of the MPTI in the native state from M. pruriens.
... [66]; IC 50 20 µM (proteasome ChT-L) [67]. Crystal structure with trypsin 1K9B [68]; ternary complex with trypsin 1D6R [62]; crystal structure with chymotrypsin 5J4Q; solution structure [69]. ...
Bowman-Birk inhibitors (BBIs) are found primarily in seeds of legumes and in cereal grains. These canonical inhibitors share a highly conserved nine-amino acids binding loop motif CTP1SXPPXC (where P1 is the inhibitory active site, while X stands for various amino acids). They are natural controllers of plants’ endogenous proteases, but they are also inhibitors of exogenous proteases present in microbials and insects. They are considered as plants’ protective agents, as their elevated levels are observed during injury, presence of pathogens, or abiotic stress, i.a. Similar properties are observed for peptides isolated from amphibians’ skin containing 11-amino acids disulfide-bridged loop CWTP1SXPPXPC. They are classified as Bowman-Birk like trypsin inhibitors (BBLTIs). These inhibitors are resistant to proteolysis and not toxic, and they are reported to be beneficial in the treatment of various pathological states. In this review, we summarize up-to-date research results regarding BBIs’ and BBLTIs’ inhibitory activity, immunomodulatory and anti-inflammatory activity, antimicrobial and insecticidal strength, as well as chemopreventive properties.
... The mechanism of the protease inhibitors is different from those discussed above in that they reduce protein digestibility through direct inhibition of the hydrolytic enzymes; as such, they are classified as the primary antinutritional factors (Krishnan, Natarajan, Oehrle, Garrett, & Darwish, 2017). The disulphide bonds present in the structure of KTI and BBI contribute to their structural integrities (Rao & Suresh, 2007;Voss et al., 1996). Thus, it is possible to deactivate the inhibitors using processing methods that cleave the disulphide linkages. ...
Background: Proteins are essential macronutrients of the human diet. Currently, major dietary sources in developed countries are of animal origin. However, the association of red meat consumption to the increased risks of
some health conditions and its unsustainable pressure on the environment have increased the interest in plant
proteins as healthier and sustainable alternatives. Of these, legumes have a great potential, but part of their proteins are indigestible due to interaction with other components such as phytate and polyphenols. As such, the
quest to improve protein accessibility has become of interest to many researchers. Germination is proposed to be
a bioprocess method to improve protein digestibility and protein biological properties.
Scope and approach: This review discusses the importance of plant proteins and the hindrance of protein digestibility. This paper also highlights the role of germination in the deactivation of antinutritional factors, hydrolysis of
indigestible proteins, and improvement of properties and content of proteins of different legume seeds.
Key findings and conclusions: Protein digestibility is dependent on the nature of antinutritional factors (e.g. trypsin
inhibitors and phytate) in the food matrix. Germination represses the activity of trypsin inhibitors and removes
the phytate-related inhibition through hydrolysis. Protein content increases in germinated seeds when compared
to non-germinated ones, suggesting that proteins were either hydrolysed or dissociated from antinutritional factors. Germination seems like an adequate bioprocessing method to improve the content and nutritional quality of
legume seed proteins.
... These results corroborate well with the reports of Norioka et al. (1982), in which peanut BBI-chymotrypsin complex prone to bind with trypsin by slow release of its partner chymotrypsin but not seen in case of BBI-trypsin complex. PnBBI also possessed excessive anti-parallel β-sheets and random coils which is a core structural feature of BBIs (Figure 6; Voss et al., 1996;Qi et al., 2005;Banerjee et al., 2017). Also, the function of PnBBI in terms of its TI and CI activity was stable at a broad spectrum of pH and temperature, and corroborated well with its unaffected Far-UV CD spectra (Figures 5A,B, 6A,B). ...
Proteinase/Protease inhibitors (PIs) from higher plants play an important role in defense and confer resistance against various insect pests and pathogens. In the present study, Bowman-Birk Inhibitor (BBI) was purified from mature seeds of an interspecific advanced hybrid peanut variety (4368-1) using chromatographic techniques. The biochemical and biophysical characteristics such as low molecular mass, presence of several isoinhibitors and higher-ordered dimer/tetramer, predominance of antiparallel β-sheets and random coils in secondary structure, reactive sites against trypsin and chymotrypsin, broad spectrum of stability toward extreme pH and temperature along with MALDI TOF-TOF analysis (ProteomeXchange identifier PXD016933) ascertained the purified biomolecule from peanut as BBI (PnBBI). Surface plasmon resonance competitive binding analysis revealed the bifunctional PnBBI is a trypsin specific inhibitor with 1:2 stoichiometry as compared to chymotrypsin. A concentration-dependent self-association tendency of PnBBI was further confirmed by ‘red shift’ in the far-UV CD spectra. Furthermore, the insecticidal potential of PnBBI against Helicoverpa armigera was assessed by in vitro assays and in vivo feeding experiments. A significant reduction in larval body weight was observed with concomitant attenuation in the activity of midgut trypsin-like proteases of H. armigera (HaTPs) fed on PnBBI supplemented diet. The one and two-dimensional zymography studies revealed the disappearance of several isoforms of HaTP upon feeding with PnBBI. qRT-PCR analysis further suggests the role of PnBBI in not only inhibiting the activity of midgut trypsin and chymotrypsin-like proteases but also in modulating their expression. Taken together, the results provide a biochemical and molecular basis for introgressed resistance in peanut interspecific advanced hybrid variety against H. armigera.
... Numerous studies have been carried out to elucidate the molecular structure of said inhibitors in an attempt to justify the mentioned behaviour. Finally, it has been reported that these inhibitors have a polar domain interface that is solvated with internal water molecules and hydrophobic patches that would be responsible for the tendency to selfassociation [61,68,69]. For all the above, we are able to affirm that the trypsin inhibitor of chañar has a molecular mass of 6743.7 Da with tendency to the formation of trimers in solution, even under the reducing conditions used in the SDS-PAGE. ...
... The crystal structure of IBB1 reveals that the protein has five disulphide-bonds exposed to the exterior and two other buried; IBB1 also shows two exposed hydrophobic patches making a very stable dimeric conformation. 26 The extremely stable structure of IBB1 with hydrogen bonds between lysine resides and other amino acids may block the contact of the glucose with the target amino acid obstructing its glycation. 27 The thermal resistance pattern of IBBD2 obtained for trypsin inhibitory activity was very different to those obtained for IBB1; a great loss of inhibitory activity was observed over time, being this loss significantly higher in the presence of glucose. ...
Naturally-occurring serine protease inhibitors of the Bowman-Birk family, particularly abundant in legume seeds, exert their potential chemopreventive and/or therapeutic properties via protease inhibition. Processing of legume seeds, including soybeans, has been proposed as a major cause for their loss of bioactivity due to glycation. In order to assess how glycation affected the protease inhibitory activities of major soybean Bowman-Birk isoinhibitors (BBI) and their antiproliferative properties, IBB1 and IBBD2 were purified and subjected to glycation under controlled conditions using glucose at high temperature. Both soybean BBI isoinhibitors showed remarkable heat stability. In the presence of glucose, IBBD2 lost most of its trypsin inhibitory activity while IBB1 maintains similar trypsin and chymotrypsin inhibitory activities as in the absence of sugar. Glycation patterns of both BBI proteins were assessed by MALDI-TOF spectrometry. Our results show that the glycation process affects IBBD2, losing partially its antiproliferative activity against HT29 colon cancer cells, while glycated-IBB1 was unaffected.
... , [4,5,7] . , BBI 8 16 kD [2,8~10] . ...
... Moreover, it forms stable binary and tertiary complexes with T and α-CT presenting binding constants from 10 7 to 10 9 M -1 [14,15]. BTCI has an unusual solvent exposed hydrophobic patch with buried hydrophilic residues at subdomain 2, a characteristic feature of BBIs that is considered the source of their self-association tendencies [13,16,17]. BTCI forms concentration dependent self-oligomers, ranging from monomers to dimers, trimers and hexamers as demonstrated by light scattering and atomic force microscopy [18,19]. ...
The black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) forms concentration dependent homomultimers, as previously demonstrated by Light scattering and Atomic Force Microscopy. Considering that these self-aggregates might influence their binding to cognate enzymes, we investigated the interaction of BTCI at picomolar concentrations using surface immobilized Chymotrypsin (α-CT) and Trypsin (T) by Surface Plasmon Resonance. Our results indicate that BTCI has subnanomolar affinity to both immobilized enzymes, which is approximately two orders of magnitude higher than previously reported. Moreover, we probed the influence of temperature on protein binding equilibria in order to investigate their interaction energetics. While the BTCI/T interaction concurs with the canonical entropy-driven mechanism described for BBI interactions with serine proteinases, the BTCI/α-CT interaction does not. Our measurements indicate that bimolecular BTCI/α-CT complexes form with a negative enthalpy change and a moderate entropic increase. Direct calorimetric evaluation is in accord with the van't Hoff approximation obtained by SPR. We demonstrate that as protein concentrations increase to the micromolar range, secondary endothermic events become prevalent and affect both the kinetics and thermodynamics of protein associations. Our study reinforces that BBI interactions with serine proteinases should be studied in dilute solutions to abridge often neglected secondary interactions.
... BBIs fold into a core of antiparallel beta-sheets that is crosslinked by multiple disulphide bridges (seven in the case of soybean BBI). The two inhibitory sites are located on protruding loops on opposing ends of the beta-sheet core ( Fig. 2a) and function via the Laskowski mechanism (Box 2) (Chen et al., 1992;Voss et al., 1996). Monocot I12 inhibitors contain up to three BBI domains (Qu et al., 2003), and thus they should in theory have up to six protease inhibitory sites. ...
Multifunctional protease inhibitors juggle jobs by targeting different enzymes and thereby often controlling more than one biological process. Here, we discuss the biological functions, mechanisms and evolution of three types of multifunctional protease inhibitors in plants. The first type is double‐headed inhibitors, which feature two inhibitory sites targeting proteases with different specificities (e.g. Bowman–Birk inhibitors) or even different hydrolases (e.g. α‐amylase/protease inhibitors preventing both early germination and seed predation). The second type consists of multidomain inhibitors which evolved by intragenic duplication and are released by processing (e.g. multicystatins and potato inhibitor II , implicated in tuber dormancy and defence, respectively). The third type consists of promiscuous inhibitory folds which resemble mouse traps that can inhibit different proteases cleaving the bait they offer (e.g. serpins, regulating cell death, and α‐macroglobulins). Understanding how multifunctional inhibitors juggle biological jobs increases our knowledge of the connections between the networks they regulate. These examples show that multifunctionality evolved independently from a remarkable diversity of molecular mechanisms that can be exploited for crop improvement and provide concepts for protein design.
Contents
Summary
794
I.
Introduction
794
II.
Three types of multifunctionality
795
III.
Significance of multifunctional protease inhibitors in the plant research arena
803
Acknowledgements
805
References
805
... The Bowman-Birk inhibitor is a monomeric protein possessing two independent subdomains for the binding of trypsin and a-chymotrypsin. The inhibitor forms a 1:1 complex with trypsin or chymotrypsin, [28] blocking their activity. First, we added the trypsin-chymotrypsin inhibitor to the solution of trypsin (1 mg mL À1 ) such that the enzyme/inhibitor molar ratio was 1:6. ...
Liquid-crystal (LC) droplet patterns are formed on a glass slide by evaporating a solution of nematic LC dissolved in heptane. In the presence of an anionic phospholipid, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG), the LCs display a dark cross pattern, indicating a homeotropic orientation. When LC patterns are incubated with an aqueous mixture of DOPG and poly-L-lysine (PLL), there is a transition in the LC pattern from a dark cross to a bright fan shape due to the electrostatic interaction between DOPG and PLL. Known to catalyze the hydrolysis of PLL into oligopeptide fragments, trypsin is preincubated with PLL, significantly decreasing the interactions between PLL and DOPG. LCs adopt a perpendicular orientation at the water–LC droplet interface, which gives rise to a dark cross pattern. This optical response of LC droplets is the basis for a quick and sensitive biosensor for trypsin.
... 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]. ...
... BBIs are induced on wounding or infection, suggesting their involvement in plant defence (Ryan, 1990). As a result of the highly stable structure of disulphide bridges (seven in dicots, five in monocots, with the exception of ORY-SAN with four; Prakash et al., 1996) and polar interactions between subdomains, BBIs are resistant to extreme conditions, such as cooking temperatures and acidic pH values present in the digestive systems of animals (Voss et al., 1996). ...
Extracellular plant peptides perform a large variety of functions, including signalling and defence. Intracellular peptides often have physiological functions or may merely be the products of general proteolysis. Plant peptides have been identified and, in part, functionally characterized through biochemical and genetic studies, which are lengthy and in some cases impractical. Peptidomics is a branch of proteomics that has been developed over the last 5 years, and has been used mainly to study neuropeptides in animals and the degradome of proteases. Peptidomics is a fast, efficient methodology that can detect minute and transient amounts of peptides and identify their post-translational modifications. This review describes known plant peptides and introduces the use of peptidomics for the detection of novel plant peptides. This review is dedicated to the memory of Clarence E. (Bud) Ryan who elegantly discovered and analysed many of the peptides described herein.
... Studies of the conformation of the inhibitor, involving NMR and crystal X-ray structure analysis, demonstrated that the active sites are located at the opposite ends of the elongated molecule (4.5 × 2.4 × 2.0 nm), at a distance of 3.6-4.0 nm from each other [93][94][95]. ...
... Another disulfide-rich protein that has interesting pharmacokinetic properties is the soybean-derived 8 kD Bowman-Birk inhibitor (BBI; Fig. 10b) [122]. BBI is not backbone-cyclized, although like the cyclotides it has a knot-like topology [123,124]. BBI suppressed radiation-induced transformation of pluripotent stem cells at sub-nM concentrations [125], and when fed to mice prevented chemicalinduced adenocarcinoma [126]. Indeed, BBI was widely distributed in tissues within 3 hours after oral administration [127]. ...
The structural complexity of many natural products sets them apart from common synthetic drugs, allowing them to access a biological target space that lies beyond the enzyme active sites and receptors targeted by conventional small molecule drugs. Naturally occuring cyclic eptides, in particular, exhibit a range of biologi -cal activities whose diversity is reflected in their rich structural variety. Many of these compounds penetrate cells by passive diffusion and some, like cyclosporine A, are clinically used, orally bioavailable drugs. These natural products tend to have molecular weights and polar group counts that put them outside the norm based on classic predictors of "drug-likeness". Because of their size and complexity, cyclic peptides can show great -er specificity and potency for biological targets compared to smaller acyclic compounds, and they may pro -vide useful scaffolds for modulating more challenging biological targets such as protein-protein interactions and allosteric binding sites. This review focuses on cyclic peptide natural products from a structural perspec -tive, in the hope of uncovering trends that might account for their unexpected pharmacokinetic behavior.
... While thiol-mediated disulfide bonds can be responsible for the stabilization of irreversible protein aggregates, they also play an important role in the stabilization of some native proteins. The soybean Bowman-Birk protease inhibitor, (BBI) with seven disulfide bridges, for example, is extremely stable against heat treatment [103][104][105]. The thermodynamic aspects of the stabilization of the native structure of globular proteins by disulfide bonds have recently been reviewed by Trivedi et al [106]. ...
The aggregation of proteins is of fundamental relevance in a number of daily phenomena, as important and diverse as blood coagulation, medical diseases, or cooking an egg in the kitchen. Colloidal food systems, in particular, are examples that have great significance for protein aggregation, not only for their importance and implications, which touches on everyday life, but also because they allow the limits of the colloidal science analogy to be tested in a much broader window of conditions, such as pH, ionic strength, concentration and temperature. Thus, studying the aggregation and self-assembly of proteins in foods challenges our understanding of these complex systems from both the molecular and statistical physics perspectives. Last but not least, food offers a unique playground to study the aggregation of proteins in three, two and one dimensions, that is to say, in the bulk, at air/water and oil/water interfaces and in protein fibrillation phenomena. In this review we will tackle this very ambitious task in order to discuss the current understanding of protein aggregation in the framework of foods, which is possibly one of the broadest contexts, yet is of tremendous daily relevance.
... c) Zeta potential monitoring of particles surface at each stage of the functionalization (pH 6.0: black; pH 8.0 grey). patches on the surface and the presence of charged amino acids and water molecules on the interior (Voss et al., 1996). This is in contrast to the majority of proteins where a hydrophobic core and exposed polar amino acids are typically observed. ...
... In addition, overexpression of specific proteases has been implicated in the progression and metastasis of various tumors. Selective control of these proteases would thus provide a therapeutic avenue for treatment [49]. A more recently discovered family of large serine proteases, the Type II transmembrane serine proteases, are membrane-bound proteins that possess an extracellular C-terminal protease domain [50][51][52]. ...
An increasingly large number of bioactive cyclic peptides have been found in nature. The enhanced biological specificity, activity, and metabolic stability of cyclopeptides, by virtue of their conformationally constrained structural feature, have attracted much attention and made these compounds extensively studied. Cyclic peptidomimetic scaffolds and templates have been widely used to assemble various spatially defined functional groups for molecular recognition and drug discovery. These efforts are complemented optimally by NMR and X-ray based structure information, and assisted by molecular modeling based structure predictive methods. Peptide cyclization also becomes an effective and commonly employed strategy for peptide modifications. During the past several decades, great effort has been made to develop more efficient methods for the synthesis of cyclic peptides and peptidomimetics, as potential drug leads and / or as models for conformational analysis. This review is aimed at highlighting novel recently developed peptide cyclization approaches, and illustrating the profitable applications.
... These proteins inhibit simultaneously two protease molecules using kinetically independent binding sites by forming a stable binary [10,11] and ternary complex [5]. BBIs are composed by two domains organized in a β-hairpin of antiparallel β-sheet121314. A new structural feature of a Bowman-Birk type inhibitor from pea seeds has recently been reported. ...
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.
... 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. As the S 2 subsite of HLE is considered to be similar to that of other mammalian Ser proteinases including PPE [26], this locus was therefore not varied. ...
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.
Plant proteinase inhibitors (PIs) are defense proteins predominantly found in storage tissues. They play defensive role against insect pests by inhibiting the activity of digestive proteases in the larval midgut. The present chapter provides a general overview of PIs including their purification, classification and basic characteristic features of each class, mechanism of action on gut proteases, and signalling pathways involved during induction of PIs under biotic/abiotic stress conditions. The rapid development of resistance in insects towards pesticides was a major limitation for the generation of pest-resistant crop plants. The role of PIs as biopesticides in the management of lepidopteran pests Helicoverpa armigera, Spodoptera litura, S. frugiperda, Achaea janata, Chilo partellus and C. suppressalis was revealed in detail. Further, the transgenic expression of PI genes alone or by pyramiding with other defense genes paved way for the development of sustainable resistance in crop plants against insect pests.
The interaction of proteinases with plant proteinase inhibitors (PPIs) has always been important in insect and plant interaction studies. Available literature suggests that proteinase inhibitors have the ability to defend plants against herbivorous insects. There are three modes of action of PPIs with proteinases: insects adapt to the presence of PPI (i) by overexpressing native proteinases, (ii) by expressing new proteinases that are resistant to the inhibitors, and (iii) by proteolytic inactivation of PIs by insect’s own midgut proteinases. Midgut proteinases have been widely studied in lepidopteran pests. In the case of Antheraea assamensis, a silk-producing lepidopteran, midgut proteinase interaction with PIs of host plants shows resistance to proteolysis by larval midgut proteases. It suggests that PPIs are stable in the alkaline environment of the gut and can potentially interact with gut proteinases. Moreover, the expression levels of PPIs differed in A. assamensis larvae when reared on different species of host plants. Pieris brassicae (euryphagous pest of crucifers) midgut proteases studies reveal that those larvae which fed on Brassica oleracea var. botrytis but not those which fed on Tropaeolum majus (garden nasturtium) adapted to ingested PPIs. These results suggest that midgut physiology of this versatile pest show considerable plasticity enabling it to feed on wide range of host plants.
Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest - Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358 ± 11 nM) and chymotrypsin (Ki of 446 ± 9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC50 = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC50 = 8 μg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.
Seed legumes have played a major role as a crop worldwide, being cultivated on about 12% to 15% of Earth's arable land; nevertheless, their use is limited by, among other things, the presence of several antinutritional factors (ANFs - naturally occurring metabolites that the plant produces to protect itself from pest attacks.) Trypsin inhibitors (TIs) are one of the most relevant ANFs because they reduce digestion and absorption of dietary proteins. Several methods have been developed in order to inactivate TIs, and of these, thermal treatments are the most commonly used. They cause loss of nutrients, affect functional properties, and require high amounts of energy. Given the above, new processes have emerged to improve the nutritional quality of legumes while trying to solve the problems caused by the use of thermal treatments. This review examines and discusses the methods developed by researchers to inactivate TI present in legumes and their effects over nutritional and functional properties.
Due to the complications of the soymilk system, the heat-induced Bowman-Birk inhibitor (BBI) inactivation mechanism is not well known. In this study, two BBI samples with low and high purities were prepared from soymilk. It was confirmed that three groups (A, C, and D) of BBI, which are contained in soybean seeds, were transferred into soymilk during processing. On heating, it was found that 1) the two subdomains of BBI were not equally heat stable, 2) the conformation of BBI gradually changed, 3) some amino acid residues (namely, cystine, serine and lysine) in BBI were degraded, 4) BBI did not tend to form intermolecular cross-links with another BBI, but did slightly with non-BBI proteins. Based on some previous studies, the conformational change of BBI was attributed to β-elimination reactions on the amino acid residues of BBI and the subsequent intramolecular reactions induced by the products yielded by the β-elimination reactions.
Soybean seeds contain three groups (A, C, and D) of Bowman-Birk inhibitors (BBI). In this study, highly purified BBI-A (approximately 96%) was obtained from soybean whey at the 0.1 g level by the complex coacervation method. BBI-A has seven disulfide bonds (SS) and no sulfhydryl group and exhibits trypsin inhibitor activity (TIA) and chymotrypsin inhibitor activity (CIA). The X-ray structure has shown that BBI-A has five exposed SS and two buried SS. Due to steric hindrance, it was reasonable to consider that dithiothreitol first attacks the five exposed SS and then the two buried SS, which was supported by the results that SS reduction with dithiothreitol could be divided into quick and slow stages, and the critical point was close to 5/7. The effects of SS reduction on TIA and CIA could be divided into three stages: when one exposed SS was reduced, both TIA and CIA decreased to approximately 60%; with increasing reduction of exposed SS, CIA gradually decreased to 8%, and TIA gradually decreased to 26%; with further reduction of buried SS, CIA gradually decreased to 2%, and TIA slightly decreased to 24%. Far-UV CD spectra showed that the secondary structure of BBI-A was slightly changed, whereas near-UV CD spectra showed that the conformation of BBI-A was substantially changed after the five exposed SS were reduced, and further reduction of buried SS affected the conformation to some extent. The results from Tricine-SDS-PAGE and C8 column showed the same trend as near-UV CD spectra. BBI-A has a structural peculiarity in that two hydrophobic patches are exposed to the exterior (in contrast to typical soluble proteins), which was attributed to the seven SS by some researchers. The results above supported the hypothesis that hydrophobic collapse of the exposed hydrophobic patches into a regular hydrophobic core occurred after the reduction of SS in BBI-A.
Complex behavior of Bowman–Birk protease inhibitor (BBI) with ι-carrageenan (LC) as a function of pH, protein to polysaccharides ratio and salt concentration was studied by turbidimetric titration, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). At fixed BBI/LC weight ratio of 5:1, turbidity and DLS results showed that pHc and pHφ1 shift to the lower pH values with the increase in ionic strength (I), whereas the former occurred at the pH lower than isoelectric point (pI = 4.2) of BBI at I ≥ 100 mM NaCl. ITC results showed that BBI binding to LC involves a two-step process with an increasing exothermic enthalpy at the first binding step. The further insight of BBI-LC complexation was studied as a function of BBI concentration, ionic strength and temperatures using ITC. The critical molar ratio (rcritical) between two binding steps was independent of protein concentration, ionic strength and temperature, although the heat flow obviously decreased with the increasing I (0–200 mM) and slightly increased with the elevated temperature (25–45 °C). The negative heat capacity (ΔCp) and the gain in nonionic contribution (ΔGno) indicated the involvement of nonelectrostatic interactions (e.g., hydrophobic effect) for the first binding step.
In this study, a simple and sensitive micro-capillary sensor for monitoring trypsin reactions using confined nematic liquid crystals (LCs) was developed. The confined LC droplet was formed through sequential injection of a LC and aqueous solution into an octyltrichlorosilane-pretreated capillary. When the confined LC droplet made contact with an aqueous mixture of phospholipids and poly-l-lysine (PLL) solutions, the electrostatic interaction between the phospholipid and PLL induced realignment of the phospholipid monolayer and produced an optical LC droplet texture of two bright lines. Trypsin can cleave peptides specifically on the carboxyl side of lysine, and in this case, trypsin activity is manifested as removal of this electrostatic interaction, leading to a change in the ordering of LC droplets confined in the capillary. Thus, the orientational transition of LC droplets results in a change in their optical response from two bright lines to a four-petal shape. Control experiments with deactivated trypsin or chymotrypsin confirmed the feasibility of this trypsin detection approach. Using the LC-based capillary sensing technique, trypsin levels as low as ~ 0.1 μg/mL could be detected. This research proposes that LC-based micro-capillary sensing systems can be utilized in the development of highly sensitive and simple sensors for detecting various enzymes.
In an earlier study (McBride JD, Freeman N, Domingo GJ, Leatherbarrow RJ. Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library. J. Mol. Biol. 1996; 259: 819-827) we described a resin-bound cyclic peptide library, constructed based on the sequence of the anti-tryptic reactive site loop of Bowman-Birk Inhibitor (BBI), a proteinase inhibitor protein. This library was used to identify re-directed chymotrypsin inhibitors with K-i values as low as 17 nM. We have now extended this work by constructing an enhanced library in which a further position, at the P-4 site of the inhibitor, has been randomized. This new library has variation at three target locations (P-4, P-1 and P-2') within the inhibitory loop region, producing 8000 variants. Screening this library allowed selection of new inhibitor sequences with K-i values as low as 3.4 nM. The success of this approach is reflected by the fact that the inhibition constant given by the selected peptide sequence is slightly lower than that reported against chymotrypsin for the most studied full length BBI protein, Soybean BBI 2-IV. Copyright (C) 2000 European Peptide Society and John Wiley & Sons, Ltd.
Protein proteinase inhibitors have been used for the treatment and prophylaxis of a variety of severe disorders, causing by uncontrollable activation of serine proteinases. Besides, use of proteinase inhibitors for improving oral bioavailability of therapeutic peptides and proteins has gained considerable scientific interest. Therefore the development of delivery systems for protein proteinase inhibitors holds great therapeutic promise. This article reviews the investigations in which the attempts to control transport pattern and biodistribution of protein proteinase inhibitors by conjugating them with appropriate carriers were undertaken. A specific concept of designing the conjugates based on the structural, physico-chemical and enzymatic aspects was suggested. It was demonstrated that the general approach is applicable to the various proteinase inhibitors, and significant and sustained specific pharmacological activity could be obtained by their conjugation.
In the present document we describe the possibility for encapsulating a water-soluble protein lacking α-helices into multilayer liposomes made of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) in order to prepare proteoliposomes. The influence of the PC/PE w/w ratio on the incorporation efficiency of the Bowman-Birk soybean proteinase inhibitor (BBI) into liposomes was studied. Increase in ionic strength did not inhibit the proteoliposome formation process. BBI encapsulation did not affect liposome sizes, surface charge and structural phospholipid organization. Confocal laser scanning microscopy showed that BBI was located in the central part of the spherical particle and between bilayers. The fluorescence intensity after anthracene-labelled phospholipids interaction with Rhodamin B-labelled BBI confirmed that the protein is deeply immersed into liposomes. The biological activity (antitrypsin and antichymotrypsin) estimation of the protein entrapped in liposomes showed that the protein active centers are spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI confirms this hypothesis and suggests that this shielding is reversible. The proteoliposomes prepared seem to be promising formulations for BBI delivery.
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.
Plant protease inhibitors (PIs) are generally small proteins which play key roles in regulation of endogenous proteases and may exhibit antifeedant, antifungal, antitumor and cytokine inducing activities. Dolichos biflorus (horse gram) is an unexploited legume, which is rich in nutrients and also has therapeutic importance. It contains a double-headed PI, which is an anti-nutritional factor. As there is no report available on its simultaneous removal and purification in single step, in this study, a double-headed PI active against both trypsin and chymotrypsin was purified from Dolichos biflorus to -14-fold with -84% recovery using an immobilized metal affinity chromatography (IMAC) medium consisting of Zn-alginate beads. The method was single-step, fast, simple, reliable and economical. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH range of 2.0-12.0 and up to a temperature of 100 degrees C for 20 min. The optimum temperature for trypsin and chymotrypsin inhibitor was observed to be 50 degrees C and 37 degrees C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC using Zn-alginate beads was useful in simultaneous purification and removal of an anti-nutritional factor from horse gram flour in single step. This procedure may also be employed for purification of other plant PIs in one step.
Rice Bowman-Birk inhibitors (RBBI), with one (8 kD) or two homologous domains (16 kD), were found to be effective trypsin
inhibitors in vitro. In this study, we demonstrate that the 25-kD protein corresponding to the three-domain RBBI indeed exists in rice in planta, and that the RBBIs are regulated by development and wounding. We also found by inhibitory activity assay that the 3:13 disulfide
bond, but not the 4:5 disulfide bond, suppresses the trypsin-inhibitory activity, and the D3 domain of RBBI3-1 has no inhibitory
activity against trypsin, chymotrypsin, paparin or subtilisin. Mutation analyses showed that conversion from Lys to Leu or
Tyr in the N-terminal P1 site in D1 domain did not create chymotrypsin-inhibitory activity, suggesting that the structure
of the reactive loop in D1 domain hinder the new inhibitory specificity at P1 site, and the chymotrypsin-inhibitory activity
might need the participation of other structures, e.g. 3:13 disulfide bond.
Soybean saponin has been reported to change the nature of proteins. Thus, crude saponin fraction or isolated soyasaponin I was added to Bowman-Birk inhibitor (BBI) from soybeans to evaluate the effect on its trypsin-inhibiting activity. The soybean saponin and soyasaponin I used in the experiment had little effect on tryptic activity. The trypsin-inhibiting activity of the mixture consisting of BBI and saponin was gradually enhanced by increasing saponin fraction or isolated soyasaponin I. Circular dichroism (CD) spectra of BBI were proportionally changed with the Increase in soyasaponin I to imply change in structure of BBI. These results suggested that the conformational change by saponin caused enhancement of the trypsin-inhibiting activity of BBI.
SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar Ki value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors.
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 seeds of the legume horsegram (Dolichos biflorus), a protein rich pulse (bean), contain multiple forms of Bowman-Birk inhibitors (protease inhibitors). The major inhibitor HGI-III contains seven interweaving disulfides and is extremely stable to high temperatures. A soluble HGI-III (rHGI) with the native N-terminus was produced using a pTWIN IMPACT(TM) purification system. Yield of rHGI was improved by introducing a trypsin sepharose affinity chromatography step resulting in ∼670 fold purification. The biochemical characteristics of rHGI point to its close similarity to seed HGI-III not only in its structure but also in its inhibitory characteristics toward bovine trypsin and chymotrypsin. The expression and purification strategy presented here promises to produce BBIs in their natural form for pharmacological and therapeutic use.
The stability of BTCI has been investigated as function of pH and temperature, following its inhibitory activity against trypsin. The isolated inhibitor of 9,084 Da is stable over pH 3 to 10 at 25 o C. BTCI showed high thermal stability ranging from 25 to 95 o C at pH 3.0 and 7.0. However, the protein lost about 20 percent of its inhibitory activity over 75 o C at pH 8.2. The results indicated that BTCI is extremely stable to heat and pH as typical of Bowman-Birk 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 decoration of aqueous interfaces of thermotropic liquid crystal with oligopeptide-based amphiphiles was investigated. The 17-amino-acid oligopeptide used in the study possessed two lysine residues and a primary amine group at the N-terminus. The polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and NHS-activated surfaces prepared by self-assembly of carboxylic acid-terminated alkanethiols on gold was used. the infrared spectrum confirmed the formation of N-succinimidyl esters by reaction of the terminal carboxylic groups with EDC/NHS. The disappearance of the peaks corresponding to the NHS groups suggested that treatment of the NHS-activated surface with the 17-amino-acid oligopeptide led to the conversion of NHS-activated carboxylic acid groups to amides. It was observed that covalent attachment of the 17-amino-acid oligopeptide to the NHS-activated carboxylic acid groups of the lipid monolayer led to an orientational ordering transition in the liquid crystal.
Plants are consumed by bacteria, fungi and animals and utilization of plant proteins requires their hydrolysis, a process which is catalyzed by proteases. Plants defend themselves against other organisms by elaborating physical and chemical defenses, the latter including protein and non-protein inhibitors of proteases. Plant protease inhibitor proteins can be either constitutive or inducible as a result of wounding or pathogen invasion. Proteases are classified into the aspartic proteases, cysteine proteases, metalloproteases and serine proteases on the basis of the involvement of aspartate, cysteine, metal ions and serine, respectively, in the catalytic mechanism. Extracellular proteases are involved in digestion, blood clotting, inflammatory responses to invasion, and extracellular matrix digestion required for angiogenesis and tissue remodelling. Intracellular proteolysis must be exquisitely regu to avoid autolysis and intracellular proteases are involved in proprotein processing, lysosome- and proteasome-mediated protein destruction, cell division and apoptosis. A large variety of plant protease inhibitor proteins and peptides have been resolved including aspartic protease inhibitor proteins, cysteine protease inhibitory phytocystatins, metallocarboxypeptidase inhibitor proteins and serine protease inhibitor proteins such as the Bowman-Birk, cereal bifunctional, Kunitz, potato type I, potato type II, mustard family, squash family, serpin and other protease inhibitors. Plant protease inhibitor proteins have potential transgenic applications for crop plant defense. A variety of non-protein protease inhibitors have also been resolved from plants. Plant protease inhibitors have potential for pharmaceutical development especially in relation to Alzheimer's disease, angiogenesis, cancer, inflammatory disease and viral and protozoal infection.
Possibility of encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures
(phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) was investigated. The influence of the PC/PE ratio (w/w) on efficiency
of incorporation of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein
encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in
the central part of the spherical particle and also between bilayers. The study of biological (antitrypsin and antichymotrypsin)
activity demonstrated partial spatial shielding of active sites of proteins entrapped in liposomes. The effect of an ionic
detergent on the activity of the encapsulated BBI and BPTI is consistent with this hypothesis and suggests that this shielding
is reversible. Stability of liposomes was examined using three various media modeling gastrointestinal fluids (gastric and
intestinal juices and fluids). Data obtained indicate that the prepared liposomes seem to be promising formulations for BBI
and BPTI delivery.
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.
Branden & Jones state, in Nature: `Protein crystallography is an exacting trade, and the results may contain errors that are difficult to identify. It is the crystallographer's responsibility to make sure that incorrect protein structures do not reach the literature.' [Branden & Jones. (1990). Nature (London), 343, 687–689.] One of several available methods of checking structures for correctness is the evaluation of atomic contacts. From an initial hypothesis that atom-atom interactions are the primary determinant of protein folding, any protein model can be tested for proper packing by the calculation of a contact quality index. The index is a measure of the agreement between the distributions of atoms around each residue fragment in the model and equivalent distributions derived from the database of known structures solved at high resolution. The better the agreement, the higher the contact quality index. This empirical test, which is independent of X-ray data, is applied to a series of successively refined crystal structures. In all cases, the model known or expected to be better (the one with the lower R-factor) has a better contact quality index, indicating that this type of contact analysis can be used as an independent quality criterion during crystallographic refinement. Modelled proteins and predicted mutant structures can also be evaluated.
The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures.
Incluye bibliografía e índice Reimpresión en 1984, 1986, 1989.
A well characterized soybean protease inhibitor, the Bowman-Birk inhibitor, has been crystallized at room temperature in the presence of polyethylene glycol 4000 by vapor diffusion against an ammonium sulfate solution containing 2-methyl-2,4-pentanediol. An x-ray diffraction study reveals that the inhibitor crystallizes in a hexagonal unit cell of symmetry P6122 (or P6522) and dimensions a = b = 91.36(2) A and c = 63.92(2) A. Each of the 12 asymmetric units contains 2 molecules of molecular weight 8000. The crystal, which diffracts barely to 3-A spacings, is fairly stable to x-irradiation and has a solvent content of approximately 52% by volume.
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.
Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.
A computer modeling procedure for assessing the stereochemical suitability of pairs of residues in proteins as potential sites for introduction of cystine disulfide crosslinks has been developed. Residue pairs with C alpha-C alpha distances of less than or equal to 6.5 A and C beta-C beta distances of less than or equal to 4.5 A are chosen for geometrical fixation of S atoms using the program MODIP. The stereochemistry of the modeled disulfides is evaluated using limits for the structural parameters of the various torsion angles and S-S bond length in the disulfide bridge. The ability of the procedure to correctly model disulfides has been checked with examples of cystine peptides of known crystal structures and 103 disulfide bridges from 25 available protein crystal structures determined at less than or equal to 2 A resolution. An analysis of results on three proteins with engineered disulfides, T4 lysozyme, dihydrofolate reductase and subtilisin, is presented. Two positions for the introduction of 'stereochemically optimal' disulfides are identified in subtilisin.
Complexes of the Bowman-Birk soybean inhibitor with either trypsin or chymotrypsin and with both of these enzymes were isolated by gel filtration. Amino acid analyses and molecular weight estimates by gel filtration were consistent with the formation of a 1:1 molar complex between inhibitor and trypsin or chymotrypsin and a 1:1:1 molar complex composed of inhibitor, trypsin, and chymotrypsin. The binary complexes were also isolated by affinity chromatography employing insoluble derivatives of trypsin and chymotrypsin. Determination of the residual enzymic activity in solutions of the isolated complexes permitted an evaluation of their dissociation constants.
Proteinase inhibitors are a class of the various dietary inhibitors of mutagenesis and carcinogenesis (Hayatsu et al., 1988). Schelp and Pongpaew (1988) have recently hypothesized that protection against cancer may result from an increase of endogenous proteinase inhibitors such as α2-macroglobulin induced by diets that are low in calories and fat. The Bowman-Birk inhibitor (BBI) of serine proteinases, a double-headed polypeptide-inhibitor of trypsin and chymotrypsin, is one of the most potent cancer chemopreventive agents (Yavelow et al., 1983, 1985). Recently, this property has been substantiated in an in vivo investigation using mice (St. Clair et al., 1990) that were exposed to dimethylhydrazine, a potent chemical carcinogen. Therefore, BBI is currently being considered as an attractive candidate for studies directed toward the prevention of several forms of cancer that are widespread in Western societies. BBI is a small single-chain polypeptide of 71 amino acids with two subdomains directed toward trypsin and chymotrypsin/elastase, respectively (Fig. 1). This protein is the prototype of a family of proteinase inhibitors occurring in legumes. The three-dimensional structure of several BBI-type proteinase inhibitors in the free form (Suzuki et al., 1987; Chen et al., 1992) and complexed with trypsin (Tsunogae et al., 1986) were published recently. The structure of BBI in solution has been determined by NMR spectroscopy (Werner and Wemmer, 1991, 1992). The inhibitory subdomains of BBI are rigidified into a polycyclic, clearly arranged and highly conserved structural framework. BBI-type proteinase inhibitors fulfill many of the criteria of an attractive model for protein engineering studies (Fersht, 1985). Presently, major efforts are devoted to the pathophysiological elucidation and pathobiochemical characterization of the role of limited proteolysis in the course of malignant transformation. However, these efforts will eventually culminate in the rational design of specific chemical agents directed toward those proteolytic enzymes that are involved in malignant transformation.
This is an account of the initial work on a protease inhibitor now generally and appropriately designated simply as BBI. It is also appropriate that this account be brief. It is the work of other investigators that provides whatever significance inhibitors of this type possess.
Protein protease inhibitors are widely distributed among different botanical families in the plant kingdom (reviewed by Liener and Kakade, 1980). Their common source is the seed but they are also present in tubers and leaves. Most of them are proteins with M
r values in the range of 8000–10,000 but there are a few notable exceptions. The inhibitors differ in specificities, most of them inhibit trypsin and many inhibit chymotrypsin. They are frequently multiheaded as a consequence of gene elongation via gene multiplication. Different kinds of inhibitors can be present in a single tissue as exemplified in barley grains, soybeans, and potato tubers (reviewed by Birk, 1987). Their presence in valuable plant foods and their possible involvement in nutritive and physiological properties have attracted the attention of nutritionists. The nutritional significance of protease inhibitors in foods has recently been discussed (Friedman, 1986). The physiological significance of plant protease inhibitors in situ has been questioned for a long time. The hypothesis that the inhibitors may have evolved as a defense mechanism of plants toward insects has been supported by various studies (reviewed by Birk, 1987). The extensively studied inhibitors of serine proteases have been classified into inhibitor families on the basis of sequence homology, assignment of the inhibitory site(s), and interaction with the protease(s) according to a standard mechanism (Laskowski and Kato, 1980; Laskowski, 1986). Since the inhibitory capacities are usually evaluated on bovine pancreatic proteases, the validity and relevance of nutritional and clinical uses of the inhibitors in other species should be questioned.
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.
The topology of a protein β-sheet, the relationship between the sequential ordering of strands and their connectedness in space, is an important and well studied feature of protein structures. The prevalent nomenclature for describing β-sheet topologies is based on following a path through the sequence order of strands and noting their separation in space. Although powerful, this approach can be usefully complemented by a notation based on following a path through the connectedness of neighbouring strands and noting sequence separation. This leads in turn to a short hand expression of sheet topology, based on a method for describing the covalent structure of small molecules, which is able to express concisely the complex non-linear topological relationships of β-sheets, including bifurcations and closed structures, in a clear and natural manner. Using this novel system of notation it is possible to follow simultaneously the sequence and hydrogen bonded connectedness of strands within the topology of a sheet.
A conventional Weissenberg camera [Weissenberg (1924). Z. Phys.23, 229–238] has been modified for macromolecular crystallography. This camera system consists of a doubly bent LiF monochromator, a newly designed Weissenberg goniometer with multi-layer-line screens and its controller. The camera is useful for recording many reflections up to high resolution on a film with high signal-to-background ratio from a crystal with large unit-cell dimensions without losing any of the diffracted beams by screens. The resulting photograph is very easy to index, and the ratio of partially recorded reflections to fully recorded reflections decreases in comparison with an oscillation camera. The multi-layer-line screens are especially practical with a continuous X-ray beam such as synchrotron radiation where both wavelength and band pass can be selected properly. The preparation of a point-focusing monochromator from a single-crystal of LiF is described, and the recording of anomalous dispersion profiles is discussed.
A new search strategy is presented to obtain initial phases for single-crystal diffraction data by molecular replacement. It consists of carrying out 'Patterson refinements' of a large number of the highest peaks of a rotation function. The target function for Patterson refinement is proportional to the negative correlation coefficient between the squared amplitudes of the observed and the calculated normalized structure factors. If the root-mean-square difference between the search model and the crystal structure is within the radius of convergence of the minimization procedure employed, the correct orientation can be identified by having the lowest value of the target function after refinement. Similar to conventional crystallographic R-factor refinement, the target function for Patterson refinement may be combined with an empirical energy function describing geometric and non-bonded interactions. Patterson refinement of individual atomic coordinates or of rigid-group parameters may be carried out. Search models of crambin and of myoglobin with 1.6-2.0 Å backbone atomic r.m.s, differences from the target crystal structures show that the Patterson refinement strategy can solve crystal structures that cannot be solved by conventional molecular replacement or even by full six-dimensional searches.
Reduction of model bias in macromolecular crystallography through various omit-map techniques has been investigated. The two cases studied were the p21 protein complexed with GDP at 2.25 angstrom resolution and the AN02 Fab fragment of an anti-dinitrophenyl-spin-label murine monoclonal antibody complexed with its hapten at 2.9 angstrom resolution. In the former case, the correct model was compared to a partially incorrect model consisting of an exchanged pair of beta strands along with rearrangement of the connecting loops whereas, in the latter case, the correct placement of an active-site tryptophan side chain was compared to an incorrect rotamer conformation. Partial structures were created by omission of spherical regions around the incorrect region. Omit maps without refinement of the partial structure showed a large degree of model bias. Model bias could be reduced significantly by refinement of the partial structure. Simulated-annealing refinement of the partial structure showed the best results, followed by conjugate-gradient minimization with or without prior randomization of the partial structure. To avoid compensation for missing atoms during simulated-annealing refinement of the partial structure, a suitable 'boundary' region was restrained to the starting coordinates. Model bias removal by iterative density modification was not successful in that it reduced density for both the correct and incorrect conformations.
A complete structural characterization in solution, by NMR spectroscopy, and in vacuo, by molecular dynamic simulations, of two synthetic peptide fragments from SBBI (Soybean Bowman–Birk Inhibitor) is reported. Peptide 197, corresponding to the SBBI(41–49) chymotrypsin recognition site, has free N- and C-terminal groups, while peptide 212, corresponding to the Leu 16-SBBI(14–22) has uncharged and fully protected terminal ends. Peptide 212 shows significant anti-chymotryptic activity while peptide 197 is inactive. Neither of the two peptides shows anti-tryptic activity. The structural information obtained in the present paper suggests a quantitative structure–activity relationship which may help both in understanding the mechanism of action of protease inhibitors, and in providing new directions for the rational design of more specific and potent inhibitors.
Retention of known geometry, with regard to mean atomic positions, has proved useful in the refinement of macromolecules. In structures with a paucity of diffraction data and large displacements of the atoms from their mean positions, it is also of value to restrain the thermal factors to be consistent with known stereochemistry. This paper presents a technique for accomplishing this by restraining the variances of the interatomic distributions (which are functions of the mean atomic positions and the thermal parameters) to suitably small values. This procedure allows meaningful anisotropic refinement of macromolecules to be carried out with low-resolution diffraction data. Anisotropic thermal parameters obtained in this way should prove useful in understanding the dynamics of the biological functions of macromolecules.
Equilibrium constants, enthalpies, and entropies of hydrogen bonding between six aliphatic alcohols and the electron donors of the title have been determined by gas-liquid chromatography in the range 30 to 60°. The equilibrium constants follow the general trend expected on the basis of alkyl group inductive and steric effects. The enthalpies are correlated within experimental error by the equation |ΔHij| = QiaQjb, where the Q's represent parameters for acid i and base j, respectively. The ratios of the base parameter values for the amine, ether, and thioether are respectively 2.02:1.48:1.00, in good agreement with previously determined nmr ratios for haloform hydrogen bonding (2.01:1.49:1.00). The enthalpies are also analyzed in terms of Drago's double-scale equation for ΔH of adduct formation. Finally, all systems follow a common linear plot of ΔH vs. ΔS.
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 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 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.
Bowman-Birk inhibitor, a major trypsin and chymotrypsin inhibitor from soybean, has 71 amino acids with 7 disulfide bonds. Conformation of Bowman-Birk inhibitor in native state, after heating, and after disulfide bonds were broken by sodium metabisulfite was determined by circular dichroism. The native Bowman-Birk inhibitor has 61% beta-sheet, 38% unordered form, 1% beta-turn, and no alpha-helical structure. There was no significant change in conformation after Bowman-Birk inhibitor was heated at 80 degrees C for 1 h in phosphate buffer. There was a decrease in beta-sheet and an increase in beta-turn structure after Bowman-Birk inhibitor was heated at 80 degrees C for 1 h in sodium metabisulfite-phosphate buffer. Although the change in conformation after disulfide bonds of Bowman-Birk inhibitor were broken was statistically significant (P < 0.05), the magnitude of the change was not large. The data support Bowman-Birk inhibitor's having a stable conformation even after disulfide bonds are broken.
Soybean Bowman-Birk inhibitor, a double-headed inhibitor of trypsin and α-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.
A polypeptide isolated from red kidney beans, , which has previously been shown to stimulate RNA synthesis in cultures of mouse spleen lymphocytes and plasmolyzed , is here shown to be a potent inhibitor of trypsin and α-chymotrypsin. This polypeptide is compared with commercially available trypsin inhibitors with regard to their capacity to inhibit some proteolytic enzymes and to stimulate cultures of lymphocytes. Similar to FV the lima been trypsin inhibitor was found to possess a stimulating effect on the RNA as well as the DNA synthesis in lymphocyte cultures.
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 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.
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.
The 1H resonance assignments and secondary structure of the trypsin/chymotrypsin Bowman-Birk inhibitor from soybeans were determined by nuclear magnetic resonance spectroscopy (NMR) at 600 MHz in an 18% acetonitrile-d3/aqueous cosolvent. Resonances from 69 of 71 amino acids were assigned sequence specifically. Residues Q11-T15 form an antiparallel beta-sheet with residues Q21-S25 in the tryptic inhibitory domain and an analogous region of antiparallel sheet forms between residues S38-A42 and Q48-V52 in the chymotryptic inhibitory domain. The inhibitory sites of each fragment (K16-S17 for trypsin, L43-S44 for chymotrypsin) are each part of a type VI like turn at one end of their respective region of the antiparallel beta-sheet. These structural elements are compared to those found in other Bowman-Birk inhibitors.
Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
The conformational characteristics of disulfide bridges in proteins have been analyzed using a dataset of 22 protein structures, available at a resolution of less than or equal to 2.0 A, containing a total of 72 disulfide crosslinks. The parameters used in the analysis include (phi, psi) values at Cys residues, bridge dihedral angles chi ss, chi i1, chi j1, chi i2, and chi j2, the distances C alpha i-C alpha j and C beta i-C beta j between the C alpha and C beta atoms of Cys(i) and Cys(j). Eight families of bridge conformations with three or more occurrences have been identified on the basis of these stereochemical parameters. The most populated family corresponds to the "left handed spiral" identified earlier by Richardson [1981) Adv. Protein Chem. 34, 167-330). Disulfide bridging across antiparallel extended strands is observed in alpha-lytic protease, crambin, and beta-trypsin and this structure is shown to be very similar to those obtained in small cystine peptides. Solvent accessible surface area calculations show that the overwhelming majority of disulfide bridges are inaccessible to solvent.
Two trypsin-like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE-cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p-aminobenzamidine (PABA)-Sepharose column, which also separated the two trypsins (TLEAff.1. and TLEAff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis of cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLEAff.1 and TLEAff.2, as determined by SDS-PAGE, were 17,000 and 24,000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLEAff.1 and 3.5 fold for TLEAff.2. Since most of the locust trypsin comprised TLEAff.2, the latter served as the main object of this study. TLEAff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5-9.0. The Km and kcat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLEAff.2. The locust trypsin was full inhibited by the proteinaceous trypsin inhibitors Bowman-Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site.
The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.
A gene coding for a Bowman-Birk-type proteinase inhibitor was synthesized chemically, cloned and expressed in Escherichia coli as a fusion protein with a ß-galactosidase fragment. The corresponding mutant inhibitor, carrying a P1= Arg16 instead of Lys and an Ile27 instead of Met was obtained after cyanogen bromide cleavage, refolding and affinity chromatography on trypsin-Sepharose.
Dissociation constants of complexes with trypsin of this mutant and wild-type Bowman-Birk inhibitor are identical within experimental error. This is explained by differential patterns of hydrogen bonds between side-chains of Arg or Lys in proteinase inhibitors and the primary specificity pocket of trypsin.
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.
Experimental conditions were established whereby it was possible to control the extent of reduction of the disulfide bonds of the Bowman-Birk soybean inhibitor (BBI) with sodium borohydride. Losses in the antitryptic and antichymotryptic activities of BBI were directly dependent on the average number of disulfide bonds which had been reduced. Both activities were completely lost upon the reduction of an average of four out of the seven disulfide bonds of BBI. Disc gel electrophoresis of partially reduced, carboxymethylated preparations of BBI revealed a complex mixture of the native and fully reduced protein as well as partially reduced intermediates present in varying proportions depending on the state of reduction. It was possible to regenerate the activity of a solution of fully reduced BBI by dilution with a disulfide–thiol redox buffer similar to that which had been employed by Saxena and Wetlaufer (Biochemistry, 9, 5015, 1970) for the reactivation of reduced lysozyme. It was concluded that BBI probably has four reducible disulfide bonds which are essential for inhibitory activity against trypsin as well as chymotrypsin.
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.
Publisher Summary This chapter deals with the recent developments regarding the description and nature of the conformation of proteins and polypeptides with special reference to the stereochemical aspects of the problem. This chapter considers the parameters that are required for an adequate description of a polypeptide chain. This chapter focuses the attention on what may be called “internal parameters”—that is, those which can be defined in terms of the relationships among atoms or units that form the building blocks of the polypeptide chains. This chapter also provides an account of the mathematical method of utilizing these parameters for calculating the coordinates of all the atoms in a suitable frame of reference, so that all the interatomic distances, and bond angles, can be calculated and their consequences worked out. This chapter observes conformations in amino acids, peptides, polypeptides, and proteins.
An analysis has been made, from the data which are currently available, of the solvent content of 116 different crystal forms of globular proteins. The fraction of the crystal volume occupied by solvent is most commonly near 43 %, but has been observed to have values from about 27 to 65%. In many cases this range will be sufficiently restrictive to enable the probable number of molecules in the crystallographic asymmetric unit to be determined directly from the molecular weight of the protein and the space group and unit cell dimensions of the crystal.