Inhibitory properties and solution structure of a potent Bowman–Birk protease inhibitor from lentil (Lens culinaris, L) seeds Enzio M Ragg1, Valerio Galbusera1, Alessio Scarafoni1, Armando Negri2, Gabriella Tedeschi2, Alessandro Consonni1, Fabio Sessa1 and Marcello Duranti1 ` Department of Agri-Food Molecular Sciences, Universita degli Studi, Milano, Italy ` Department of Animal Pathology, Hygiene and Veterinary Public Health-Section of Biochemistry, Universita degli Studi, Milano, Italy Keywords Bowman–Birk inhibitor; antitryptic activity; dicotyledonous plant; Lens culinaris; nuclear magnetic resonance Correspondence E M Ragg, Department of Agri-Food ` Molecular Sciences, Universita degli Studi, via Celoria 2, 20133 Milano, Italy Fax: +39 0250316801 Tel: +39 0250316800 E-mail: enzio.ragg@unimi.it (Received 10 May 2006, revised 29 June 2006, accepted July 2006) doi:10.1111/j.1742-4658.2006.05406.x Bowman–Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed In this work we present data on the isolation of a lentil (Lens culinaris, L., var Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization LCTI is a 7448 Da double-headed trypsin ⁄ chymotrypsin inhibitor with dissociation constants equal to 0.54 nm and 7.25 nm for the two proteases, respectively The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme This is due to a substantial difference in the kon and k*on values (1.1 lm)1Ỉs)1 vs 0.002 lm)1Ỉs)1), respectively, for the intact and modified inhibitor A similar behaviour was not observed with chymotrypsin The twenty best NMR structures concurrently showed a canonical Bowman–Birk inhibitor (BBI) conformation with two antipodal b-hairpins containing the inhibitory domains The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10-Asp26-Arg28 and Asp36-Asp52 residues At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C-terminus, and a highly positive potential, surrounding the antitryptic domain The segment 53–55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman–Birk inhibitor family Bowman–Birk inhibitor (BBI) proteins are serine protease inhibitors First isolated from soybean seeds by Bowman [1] and subsequently characterized by Birk et al [2], BBIs are found in several plant sources, specially mono- and dicotyledonous seeds [3] BBIs from dicots usually have a molecular mass of 7–8 kDa and are double-headed serine protease inhibitors, while those from monocots are more variable both in size and inhibitory sites Like many other cotyledonary proteins, BBIs are the products of a multigene family within the same species [4–6] and consequently several isoforms have been Abbreviations BApNA, N-benzoyl-DL-arginine-p-nitroanilide; BBI, Bowman–Birk inhibitor; COSY-DQf, two-dimensional correlation spectroscopy doublequantum filtered; C.S.I., chemical shift index; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt; GPpNA, N-glutaryl-L-phenylalanine-pnitroanilide; LCTI, Lens culinaris trypsin inhibitor; LCTI*, Lens culinaris trypsin inhibitor hydrolysed form; MD, molecular dynamics; MSTI, Medicago scutellata trypsin inhibitor; PSTI-IVb, Pisum sativum trypsin inhibitor isoform IVb; SA, simulated annealing; sBBI, soybean Bowman–Birk inhibitor; SFTI, sunflower trypsin inhibitor 4024 FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS E M Ragg et al identified [7,8] Despite their pronounced microheterogeneity, BBIs share a relatively high degree of sequence homology, especially in the inhibitory domains, and a highly conserved disulphide bridge network [9], forming a consensus motif (Prosite code: PDOC00253) There have been various hypotheses on the physiological function of BBIs, including defence and protection, developmentally regulatory and sulphur-storage roles, with no conclusive definition as yet [10] Plant cell biology data on BBIs biosynthesis and translocation to the secretory pathway are also missing From the inhibitory viewpoint most BBIs, especially those from dicotyledonous seeds, have a doubleheaded structure bearing two independent proteinase binding sites, often one trypsin and one chymotrypsin domain Various synthetic peptides consisting of a single inhibitory domain and bearing the inhibitory activity have been produced and this has served to identify the role of specific amino acid residues in the proteinase inhibition [11] The renewed interest for this class of protease inhibitors [12] is mainly based on the findings that BBIs may act as cancer preventive and suppressing agents in a wide variety of in vitro and in vivo model systems [13] In some cases, as in the treatment of oral leukoplakia lesions, the use of BBIs has reached phase II of clinical trials [14,15] Besides the anticarcinogenic effects, BBIs also showed anti-inflammatory activity, by inhibiting the inflammation-mediating proteases [16] More recently, a number of patents on the use of BBIs against various apparently unrelated diseases have appeared [17–19] The molecular basis of these BBI activities has not been established so far, however, because a high protease activity has been shown to be connected with tumour formation and other diseases associated with angiogenesis; it has been suggested that the chemopreventive action might be related to the protease, especially antichymotrypsin, inhibitory activity [20] There has been more and more research into the involvement of specific food proteins and peptides as causative agents in the prevention and control of various diseases, many of which are related to the Western lifestyle, such as obesity, diabetes and cardiovascular diseases Furthermore, the search for novel biologically active protein molecules and their exploitation as drugs or nutraceutical agents imply their functional and structural characterization Based on these considerations, the identification of novel BBI inhibitors, either as natural compounds or synthetic peptides, and the elucidation of their structural and functional properties, is extremely important A recent review dealt with legume-derived inhibitors [21] Inhibitory properties and NMR structure of a lentil BBI We present here our results on the isolation, functional and structural analysis of a BBI from lentil (Lens culinaris L var Macrosperma) seeds Our isolation procedure yielded a protein in sufficient amounts and purity to obtain the complete amino acid sequence and 1H-NMR chemical shift assignment, as well as the measurement of interproton distances, by means of homonuclear correlation and nuclear Overhauser effect experiments The experimental values were then applied as restraints for molecular dynamics calculations leading to the three-dimensional solution structure of the protein Kinetic studies have shown that the isolated BBI from Lens culinaris seeds (Lens culinaris trypsin inhibitor; LCTI) is characterized by unusual inhibitory properties within the family of natural Bowman–Birk inhibitors Results Purification, mass spectrometry analysis and primary structure determination of LCTI The purification of LCTI from lentil seeds involved various chromatographic steps, including a final affinity chromatography step on agarose-immobilized trypsin The antitrypsin activity was measured at every purification step by N-benzoyl-dl-arginine-p-nitroanilide (BApNA) hydrolysis assays Purity was greater than 98%, as proved by RP-HPLC and SDS ⁄ PAGE (not shown) The final product was characterized by N-terminal amino acid sequencing, mass spectrometry (MALDI-TOF) (Fig 1), amino acid sequence analysis of Lys-C generated fragments and 1H-NMR The isolated 67 amino acid protein had the same primary structure as a recently published BBI, named LCI1.7, extracted from Lens culinaris var Microsperma [22], with the exception of a C-terminal missing glutamic acid residue (SwissProt Acc No Q8W4Y8) The molecular mass calculated from the primary structure (7448.29 Da assuming seven disulfide bonds) agrees with the one determined by mass spectrometry (7446.63 Da) In the amino acid sequence (Fig 2), several characteristic regions could be identified, including 14 Cys residues and the consensus sequences CTR(K)SxPPTC and CxY(L ⁄ R)SxPxQ(K)C for the antitrypsin and antichymotrypsin sites, respectively [5] Figure shows the amino acid sequence alignment of LCTI with other inhibitors of the Leguminosae family of known 3D structure Sequence identity of lentil BBI ranged from a minimum of 47% with Lima bean BBI to a maximum of 82% with pea BBI Major differences are located at the N- and C-termini Identities or conservative substitutions were observed at the inhibition FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS 4025 Inhibitory properties and NMR structure of a lentil BBI E M Ragg et al Fig Hydrolysis of 213 lM BApNa as function of time in the presence of 0.1 lM trypsin (pH 8.2, 37 °C) and the following amounts of LCTI: lM (¯, curve 0), 0.038 lM (*, curve 1), 0.11 lM (·, curve 2), 0.225 lM (h, curve 3) Fig MALDI-TOF mass spectrum of LCTI Sin: sinapinic acid The insert shows an expansion of the molecular peak sites, with the only exception being Medicago scutellata BBI, which, because it is a double trypsin inhibitor [23], has an arginine residue instead of a tyrosine or leucine in the position P1 of the antichymotryptic site (P and P¢ nomenclature according to Schechter and Berger [24]) Antitrypsin and antichymotrypsin activity assays The inhibitory activity of LCTI was determined at pH 8.2, by monitoring the hydrolysis of the chromogenic substrates BApNA and N-glutaryl-l-phenylalanine-p-nitroanilide (GPpNA) in the presence of bovine trypsin and a-chymotrypsin, respectively, and increasing amounts of LCTI Figure reports the amount of hydrolysed BApNA as a function of time In the presence of LCTI, two distinct kinetic regimes with different rate constants were present This effect was more evident for equimolar LCTI ⁄ trypsin mixtures, whereas in the case of low amounts of LCTI the first kinetic phase vanished after a few minutes of the reaction N-terminal amino acid sequencing of the proteolytic fragments (see below) proved that hydrolysis actually occurred in the antitrypsin site at the cleavable N-terminal P1-P1¢ bond (not shown) The kinetic model assumed (Scheme 1) implies the formation of a : complex [25] and is the simplest one able to fit with sufficient accuracy the experimental results The kcat ⁄ KM ratio was derived by fitting the experimental data in the absence of inhibitor and agreed with kcat and KM independently determined by means of standard Lineweaver–Burk analysis At [LCTI] ⁄ [trypsin] ¼ 0.38, as LCTI is hydrolysed within a few minutes (Fig 3, curve 1), its inhibitory activity is mainly due to Lens culinaris trypsin inhibitor hydro- Scheme Fig Sequence alignment of LCTI with other inhibitors from Leguminosae family of known 3D structure Accession numbers are from Brookhaven Protein Data Bank and refer to the following proteins: LCTI (2AIH_lens, this work); MSTI (1MVZ_Medicago); PSTI-IVb (1PBI_ pea); sBBI (1BBI_soya); lima bean trypsin inhibitor (LBTI) (1H34_lima) T and CT denote P1 residues in the antitrypsin and antichymotrypsin sites, respectively 4026 FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS E M Ragg et al Inhibitory properties and NMR structure of a lentil BBI lysed form (LCTI*), leading to an accurate measurement of k*on and k*off At [LCTI] ⁄ [trypsin] ¼ 1.1 and 2.25 the conversion of LCTI into LCTI* (Fig 3, curves and 3), allowed the simultaneous computation of koff, k*on and k*off The kon value was assumed by analogy with soybean BBI [26] The two dissociation constants (Kd ¼ koff ⁄ kon and K*d ¼ k*off ⁄ k*on, relative to the virgin and modified inhibitor, respectively) were calculated on the basis of the derived kinetic constants The results, obtained after simultaneous fitting of all the experimental curves, are reported in Table The same type of kinetic analysis was applied for assaying the antichymotryptic activity In analogy to the previous experiment, a partial loss of chymotrypsin inhibitory activity was observed, but it was less evident due to a lower rate of hydrolysis and, more importantly, to a minor difference between Kd and K*d (Table 1) H-NMR sequential assignments and secondary structure determination A total of 62 NH-Ha interactions were detected through the analysis of the TOCSY and two-dimensional correlation spectroscopy double-quantum filtered (COSY-DQf) experiments, allowing the identification of the characteristic amino acid spin systems The arginine residues were identified through the connectivities with their e-NH protons Two amide protons, belonging to spin systems of the type NHb CHa-CH2 and later identified as Asp10 and Asp36, were detected at very low field (11.48–11.49 p.p.m.) Sequential assignments were performed using well established procedures [27] on the basis of the dNN(i,i+1) and daN(i,i+1) interactions observed in the NOESY experiments Other weak connectivities were detected in the TOCSY and NOESY spectra, where the sequential assignment pathway between residues 12 and 16 was found split in two, thus suggesting that a minor form of LCTI (% 10%) was present in the solution As residue 16 is located in the antitrypsin site, this form was attributed to LCTI* Additional resonances, attributed to LCTI*, were found for Thr53 and His54 This finding is consistent with the presence of a minor peak in the mass spectra, which corresponds to a mass increase of 18 Da, as expected from Fig LCTI b-hairpin elements (segments Thr11-Val25 and Lys37Tyr51), with observed NOE interactions (double-arrow) and hydrogen bonds involving the slowly exchanging amide protons (dotted line) T and CT denote the antitrypsin and antichymotrypsin sites, respectively the hydrolysis of one peptide bond (Fig 1, insert) Moreover, minor peaks corresponding to the sequence starting with Ser17 were detected in the previously mentioned amino acid sequence analysis (not shown) Indeed, both NMR and MS spectra showed that the amount of hydrolysed form increased when the inhibitor was kept in solution at pH 3.1 for few days, suggesting a particular intrinsic lability of the Arg16Ser17 bond to hydrolysis at acidic pH The sequential inter-residue interactions provided a means for defining the cis-trans conformation for the two pairs of contiguous prolines Thus, Pro20 and Pro46 were found in trans-conformation, because of the strong Pro19Ha-Pro20Hd and Pro45Ha-Pro46Hd interactions, whereas Pro19 and Pro45 were classified as cis by means of the detected sequential daa(i,i+1) interactions, respectively, with Gln18 and Asn44 No d(i,i+3) interaction was observed, thus excluding the presence of any helical segment or type-I ⁄ II turn, within the protein Figure reports the relevant sequential NOE interactions for the two inhibitory regions, located in the Thr11-Val25 and Lys37-Tyr51 segments They are characterized by clusters of strong daN(i,i+1) and weak daN(i,i+1) interactions and, Table Kinetic and thermodynamic parameters for the inhibitory activity of LCTI against bovine trypsin (BT) and a-chymotrypsin (BCT), measured at pH 8.2 kon · 10)6 values taken from [26] kon · 10)6 (M)1Ỉs)1) BT BCT koff · 103 (s)1) k* · 10)3 (M)1Ỉs)1) k*off · 103 (s)1) K 1.1 0.2 0.60 ± 0.15 1.45 ± 0.21 2.0 ± 0.8 12.5 ± 5.0 0.82 ± 0.01 0.40 ± 0.05 0.54 ± 0.1 7.25 ± 1.08 FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS d · 109 (M) K*d · 109 (M) Khyd 410 ± 95 32 ± 13 759 4.4 4027 Inhibitory properties and NMR structure of a lentil BBI E M Ragg et al together with several detected long-range daa and daN interactions, define two b-hairpin secondary structure elements Figure 5A reports the chemical shift index (C.S.I.) for Ha, in comparison with the corresponding soybean BBI values Random coil values were taken from [28] Positive values indicate a residue propensity for extended or b-sheet structure [29] Thus, the C.S.I analysis identifies six b-sheet regions The LCTI data are very similar to soybean, with the exception of the 26–29 segment, with positive C.S.I values more similar to Medicago scutellata trypsin inhibitor (MSTI) [23] and the 49–55 segment, characterized by a marked reduction in propensity for an extended conformation At the end of the antitrypsin and antichymotrypsin b-hairpin, the segments Thr21-Cys22 and Gln47-Gln49 experience long-range interactions, respectively, with the segment Thr53-Lys55 and Arg28-Glu29 In these cases, however, the pattern of the observed NOE inter- actions is not sufficient to indicate the presence of additional b-strands, but rather a spatial proximity of these short segments to the b-hairpins Measured values of the vicinal coupling constants provided additional restraints for the corresponding dihedral angles, to be introduced in the restrained molecular mechanics and dynamics calculations The N- and C-terminus segments appeared rather structureless, with no detected long-range NOE up to the Cys8-Cys61 disulphide bond Deuterium exchange experiments and temperature coefficient measurements The analysis of the secondary structure suggested the presence of several hydrogen bonded amide protons, mainly located near the two inhibitory sites Deuterium exchange experiments were thus performed, by directly dissolving the protein in D2O and acquiring a series of one-dimensional spectra at room temperature After a few hours after dissolution, 11 amide protons were still observable and could easily be assigned In order to fully characterize the solvent accessibility of the amide protons, the chemical-shift temperature coefficients (DdNH ⁄ DT) were determined by performing a series of TOCSY experiments at various temperatures (Table 2) As absolute values less than p.p.b.ỈK)1 indicate solvent protection, the temperature coefficients are a complementary measurement for the more direct deuterium-exchange experiments and are particularly suitable for amide protons in the fast-exchange regime The analysis of the experimentally determined values, and their implication with the peptide tertiary structure, will be discussed below Solution structure of LCTI Fig (A) Comparison of C.S.I values for LCTI (white) and soybean BBI (black) calculated with 3-point smoothing Data for soybean BBI were taken from Biological Magnetic Resonance Data Bank (Acc no 1495); (B) Local rmsd values calculated from the superimposition of the 20 NMR-derived structures b-hairpin regions are underlined 4028 The observed NOEs also provided information on the global protein folding All the measured vicinal coupling constants and NOE interactions were translated into restraints for the generation of the solution structure Statistics for the total amount of experimental data are reported in Table A simulated annealing (SA) procedure was used starting from a randomly generated linear polypeptide chain The actual protocol is described in detail below Initially, no disulphide bond definition was introduced and a limited subset of distances, derived from NOESY experiments performed at short mixing times (tmix ¼ 80 ms), was utilized for generating a starting restraints set, together with ideal values for /,w dihedral angles One hundred and fifty structures were thus obtained and analysed in terms of total energy, FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS E M Ragg et al Inhibitory properties and NMR structure of a lentil BBI Table Temperature coefficients (Dd ⁄ DT) and deuterium exchange rates (kex) of LCTI amide protons Estimated accuracy of temperature coefficient is ± 0.1 p.p.b.ỈK)1 Residue Dd ⁄ DT (p.p.b.ỈK)1) kex · 103 (min)1) Residue Dd ⁄ DT (p.p.b.ỈK)1) kex · 103 (min)1) D2 D3 V4 K5 S6 A7 C8 C9 D10 T11 C12 L13 C14 T15 R16 S17 Q18 T21 C22 R23 C24 V25 D26 V27 R28 E29 S30 C31 H32 S33 A34 )6.1 )6.5 )7.8 )7.6 )5.8 )6.8 )4.0 )5.1 )5.8 )5.8 )8.8 )4.4 )8.5 )7.8 )7.4 )1.2 )9.4 )2.4 )4.8 )2.7 )3.7 )4.5 )1.7 )8.7 )4.1 )6.8 )2.4 )8.5 )12.7 )11.4 )5.0 – > < < > – > > > < – < < > > > – < < < < < > – < – < – – > – C35 D36 K37 C38 V39 C40 A41 Y42 S43 N44 Q47 C48 Q49 C50 Y51 D52 T53 H54 K55 F56 C57 Y58 K59 A60 C61 H62 N63 S64 E65 I66 E67 )4.4 )5.8 )2.1 )10.0 )5.1 )9.4 )3.4 )6.5 )4.8 )10.0 )6.5 )8.0 )1.2 )3.8 )5.7 )0.1 )7.5 )2.1 )8.8 – )7.5 )12.7 )7.5 )8.4 )2.2 )7.4 )7.2 )8.0 )3.1 )7.4 )8.2 < > < – < – < > – – < < < < < – – < – – – > – < > – > – – < > 600 40 40 600 600 600 600 3 40 600 600 600 40 40 600 40 600 600 600 3 600 600 40 40 600 600 40 600 600 40 600 restraint violations and chirality for Ca atoms From the initial set, a family of 55 structures was extracted with consistent folding topology and disulphide bonds The selected structures where refined by another restrained SA step, starting at 100K, and final minimization In order to reduce the overall molecular charge, during refinement 11 chloride ions were introduced at random positions, after protonation of all side chains (the NMR experiments were all performed at pH 3.1), introduction of a layer of water molecules and switching the force-field to Charmm22 Vicinal coupling constants for amide protons and the full set of NOESY cross-peak volumes were finally introduced in place of the previous dihedral angles and interproton distance restraints Assuming isotropic motions, an overall correlation time value of ± ns was found This value is consistent with the presence of monomeric species Indeed, under these conditions the protein was found to adopt a monomeric form, as assessed by size exclusion chromatography on Superdex peptide-HPLC and DOSY experiments The best 20 structures were selected on the basis of Ramachandran plot quality [30]: for this subset of structures 64.1% amino acid residues were in the most favoured region; 31.6% in the allowed one; 4.3% in the generously allowed one No amino acid was found in the disallowed region Figure reports the superimposed Ca chains of the NMR-derived structures The calculated rmsd values for selected regions are reported in Table 3, as well as the statistics of the considered structures and the relevant conformational energy parameters Figure 5B shows the local rmsd values calculated with a five-residue window As judged by the reported rmsd values, the two inhibitory sites consist of fairly rigid secondary structure elements, connected by segments with augmented conformational mobility The antitrypsin domain comprises the 11–25 segment, incorporating an antiparallel b-sheet (amino acids 11–15 and 21–25) and a type-VIb b-turn For this region, the calculated rmsd ˚ value within the deposited structures is 0.62 A (Table 3) The conformation of the 16–20 region is mainly defined by the two vicinal prolines (Pro19 and Pro20), found, respectively, in the cis- and trans-conformation, as previously discussed The corresponding /,w-values are reported in Table in comparison with those obtained from the X-ray structure of Pisum sativum trypsin inhibitor isoform IVb (PSTI-IVb) [31] Folding similarities of LCTI with PSTI-IVb and soybean Bowman–Birk inhibitor (sBBI) are shown in Fig 7, reporting the superimposition of Ca carbons ˚ (rmsd 1.99 and 2.10 A, respectively, calculated considering the peptide region within the Cys8-Cys61 bond) Some conformational heterogeneity of LCTI around the scissile bond is present, as two conformations were actually found at the level of Arg16-Ser17, one being similar to the one of PSTI-IVb The b-hairpin motif is stabilized by a hydrogen bond network connecting Thr11-Val25 and Leu13-Arg23 pairs The presence of such hydrogen bonds is proved also by the chemical shift temperature coefficients (Dd ⁄ DT < p.p.b.ỈK)1) and the very slow solvent exchange rates of the corresponding amide protons (kex < · 10)3 min)1) Thr21NH is also characterized by a low value of chemical shift temperature coefficient and slow exchange rate All other amide protons residing between Thr15 and Gln18 are solvent exposed The amide protons of Cys22 and Cys24, with low chemical temperature coefficients, are not fully exposed to solvent This indicates that these residues are involved in other tertiary interactions, in particular with the 52–55 segment A spatial FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS 4029 Inhibitory properties and NMR structure of a lentil BBI E M Ragg et al Table Statistics for the 20 best structures derived from the restrained MD calculations rmsd, root mean square deviation Conformational energy parameters Bonds Dihedrals Impropers Angles Van der Waals Electrostatic Total Energy E (kcalỈmol)1) 41 356 15 201 )184 )225 204 ± ± ± ± ± ± ± 13 21 12 32 53 Deviations from average ‘‘topallhdg’’ structures (rmsd)bb (6–61) (rmsd)heavy (6–61) ‘‘Charmm22’’ structures (rmsd)bb (6–61) (rmsd)heavy (6–61) (rmsd)bb (11–25) (rmsd)bb (37–51) ˚ 0.90 A ˚ 1.63 A 1.08 1.88 0.62 0.64 ˚ A ˚ A ˚ A ˚ A Ramachandran plot statistics Amino Amino Amino Amino acids acids acids acids in in in in most favoured region allowed region generously allowed region disallowed region 64.1% 31.6% 4.3% 0% Number of restraints di,i NOESY intensities di,i+1 NOESY intensities di,i+2 NOESY intensities di,i+n NOESY intensities Distance restraints H-bond restraints Coupling constants (3JNa) 1099 654 49 266 632 29 Restraint deviations Distances ˚ dij > 0.9 A ˚ dij > 0.5 A H-bond ˚ dij > 0.3 A ˚ dij > 0.1 A Coupling constants JaN > 0.10 Hz JaN > 0.14 Hz 29 10 Restraint deviations (rmsd) Distance restraints H-bonds JaN R-factor 4030 ˚ 0.21 ± 0.020 A ˚ 0.024 ± 0.006 A 0.086 ± 0.011 Hz 0.095 ± 0.002 interaction actually exists between Thr21 and Thr53 methyl groups and between Cys22 and the Thr53Lys55 segment This latter segment is oriented perpendicular to the average plane of the antitrypsin domain and presents an extended but loose conformation, as judged by the fast water exchange of His54-NH (kex < 600 · 10)3 min)1), which, in an ideal b-sheet structure should be hydrogen bonded with Cys22-CO The greater conformational mobility, with respect to the antitrypsin b-hairpin, is substantiated by JNa coupling values of 5.5 Hz, by the measured low chemical shift indexes and by the calculated rmsd value, increas˚ ing up to 0.8 A just at the level of His54 (Fig 5B) The antichymotrypsin domain adopts in a similar way a b-hairpin structure, comprising a type-VIb b-turn (Table 4) within the 37–51 segment (rmsd ¼ ˚ 0.64 A) and lying on a plane almost perpendicular to that of the antitrypsin domain The hydrogen bond pattern involves Tyr51-NH ⁄ Lys37-CO, Gln49NH ⁄ Val39-CO and Gln47-NH ⁄ Ala41-CO pairs (Fig 4) The extent of the b-hairpin is defined by Gln47 and Tyr51, which, together with the amide protons located in the middle of the b-sheet structure, exchange very slowly with solvent and display low chemical shift temperature coefficients (Table 2) The hydrogen bond partner of Gln47 is Ala41, with Dd ⁄ DT and kex values lower than the dyad related Thr15 In contrast to His54 in the antitrypsin domain, Arg28NH forms a strong hydrogen bond with Cys48-CO (kex < · 10)3 min)1) The observed Arg28-NH ⁄ Cys48-CO interaction is supported also by long range NOEs (Val27-Ha ⁄ Cys48-NH and Arg28-NH ⁄ Cys48NH) The chemical shift index for the 27–29 segment indicates a propensity to adopt an extended structure (Fig 5A) The r.m.s.d values (Fig 5B) are lower than the dyad-related segment 53–55, as well as the kex values and temperature coefficients of the amide protons, suggesting a closer interaction with the antichymotryptic domain for the 27–29 segment The JNa values measured for the Val27-Arg28-Glu29 segment (5.72 Hz, 5.68 Hz, and 3.12 Hz, respectively) indicate, however, that a certain degree of local conformational mobility is still retained up to Glu29, where the peptide backbone folds into a sharp turn Relevant hydrogen bonds were found between Asp10-NH ⁄ Asp26-COOH and Asp36-NH ⁄ Asp52COOH residue pairs (Fig 8) The same hydrogen bonds are found in PSTI-IVb [31] Both Asp10 and Asp36, related by the pseudo-dyad axis, have their amide protons unusually low-field shifted and, as judged by their very fast solvent exchange rates, are solvent exposed This feature is relative to positions 10 and 36 only and is common to the other BBIs, whose FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS E M Ragg et al Inhibitory properties and NMR structure of a lentil BBI Fig Superimposition of the best 10 LCTI structures derived from restrained simulated annealing calculations Ca atoms only are displayed Table Conformational parameters for the 15–21 and 21–47 regions of LCTI Averaged /,w-values derived from the 20 NMR structures in comparison with PSTI X-ray data LCTI data from this work PSTI-IVb data taken from [31] Residue no 15 16 17 18 19 20 21 41 42 43 44 45 46 47 LCTI / (°) )81.4 ± 5.8 )81.7 ± 0.1 )176.8 )63.2 )155.9 )67.9 )71.6 )151.5 )77.6 )83.7 )60.3 )143.1 )68.7 )68.6 )123.7 PSTI-IVb / (°) w (°) ± ± ± ± ± ± ± ± ± ± ± ± ± 0.1 0.2 0.3 2.4 2.6 24.4 32.6 0.3 0.5 17.8 2.0 2.7 12.1 w (°) 149.3 115.1 18.4 )170.6 ± ± ± ± 9.3 6.6 9.2 9.2 )75.3 )84.0 164.7 )4.3 )57.5 178.7 101.5 156.7 165.0 )168.1 142.5 22.5 )163.0 105.1 162.1 154.5 121.6 ± ± ± ± ± ± ± ± ± ± ± 5.9 6.5 15.1 12.9 6.7 18.8 14.8 6.4 8.0 5.7 33.7 )149.5 )70.5 )75.9 )115.6 )95.7 )87.4 )120.4 )120.9 )74.8 )65.3 )130.3 111.9 149.4 157.4 131.5 150.0 58.7 169.5 119.7 162.4 162.4 114.8 H-NMR spectra have been assigned, i.e sBBI [32] and MSTI [23] However, Asp36 is not a highly conserved residue, because in MSTI and sBBI a lysine is present at that position, whereas PSTI-IVb has a leucine Despite this residue heterogeneity, a remarkable similarity in the corresponding amide chemical shifts exists; thus, the origin of the two unusual low-field shifts should be localized in the hydrogen bonded partners, the highly conserved Asp26 and Asp52 residing, respectively, at the end of the antitrypsin and antichymotrypsin b-hairpin domains Asp26 side chain is also involved in an additional ion pair (Fig 8A) with Arg28 (present in MSTI and sBBI but not in PSTI), whose amide proton forms a strong hydrogen bond with Cys48-CO on the antichymotryptic domain Indeed, the wealth of existing polar interactions provides high thermal stability and restricted conformational mobility for this protein region The residue dyad-related to Arg28 is His54, which should be unable to form ion pairs with Asp52 and Asp36 at neutral pH, and whose amide proton does not form, as previously discussed, a strong hydrogen bond with Cys22-CO in the antitrypsin domain Another potential hydrogen bond acceptor of His54 side chain might be Ala34-CO (Fig 8B), but this interaction is not a constant feature for all deposited structures, due to a high mobility of the histidine side chain The final structure results in an asymmetric distribution of opposite charges, at pH values around neutrality (Fig 9) The electrostatic potential is unevenly distributed on the protein surface, as a negative potential is calculated at the C-terminus, near the antichymotryptic site, whereas the antitryptic domain is highly positive due to a cluster of charged residues This might suggest a possible dimerization in solution at neutral pH values, as described for other BBIs [33] In particular, the prerequisite indicated for BBI dimerization, consisting in the unique interaction between Arg ⁄ Lys at P1 of the first BBI subunit and Asp ⁄ Glu at the carboxyl-terminus of the second subunit, is also fulfilled by LCTI Discussion This work reports the purification, primary structure analysis, kinetic properties and solution structure of a FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS 4031 Inhibitory properties and NMR structure of a lentil BBI E M Ragg et al Fig Superimposition of the LCTI solution structure (PDB ID 2AIH, red) with PSTI-IVb (PDB ID 1PBI, green) and sBBI (PDB ID 1BBI, blue) Ca atoms only are displayed Fig Electrostatic interactions and hydrogen bond networks determined in the solution structure of LCTI: Asp10-Asp26-Arg28 triad (A); Asp36-Asp52 and His54-Ala34 residue pairs (B) trypsin ⁄ chymotrypsin Bowman–Birk inhibitor isolated from lentil seeds The polypeptide, consisting of 67 amino acid residues and having a molecular mass of 7448 Da, clearly belongs to the wide family of dicotyledonous BBIs on the basis of its characteristic primary structure with a conserved Cys consensus pattern The protein is coded by the Lens gene class F1-R1, as depicted by Sonnante et al [8] As previously mentioned, lentil seeds contain various BBI isoforms which are the products of few genes; however, only one specific form has been used for the kinetic and structural analyses carried out in this work LCTI is one of the most potent natural Bowman– Birk inhibitors [34,35], with measured inhibitory 4032 parameters (Kd) against trypsin and chymotrypsin, respectively, equal to 0.54 nm and 7.25 nm As with many other BBIs, LCTI is cleaved specifically at the P1-P1¢ bond by trypsin The hydrolysed form is, however, characterized by a two orders of magnitude weaker affinity for trypsin, leading to a K*d ⁄ Kd ratio (termed Khyd) very far from unity, notably the reference value established for canonical BBIs [36,37] Thus, the measured trypsin-inhibitory activity of LCTI reflects a behaviour generally observed in synthetic peptide mimics [34] and is rather unusual for a Bowman–Birk inhibitor isolated from a natural source By contrast, the kinetic and thermodynamic parameters, derived for the antichymotryptic activity of LCTI, FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS E M Ragg et al Inhibitory properties and NMR structure of a lentil BBI Fig Particle mesh Ewald electrostatic potential calculated for LCTI at pH Isopotential curves are displayed at )60 kTỈe)1 (red) and at +60 kTỈe)1 units (blue) reside within the framework of the established general behaviour, as the determined Khyd value of 4.4 is fully consistent with the predictions based upon the dependence of such a parameter on pH [38] For some inhibitors, complex formation is a twostep process [39], involving the rapid formation of a loose complex, which slowly evolves into a more tightly bound one The slow formation of a more stable complex would, however, lead to an apparent increase in inhibitory activity This is the opposite of what we actually observed The experimental design of the inhibitory activity assays for this class of inhibitors is thus particularly important [40], within the context of structure–activity relationship studies, as the marked loss of inhibitory activity due to fast hydrolysis might lead to the determination of apparent lower affinities, if not properly measured This observation might explain the measured Kd value of 7.9 nm for a previously identified BBI from Lens culinaris (LCI-1.7) [41], a value likely to correspond to an LCTI ⁄ LCTI* mixture In our case, the initial presence of LCTI* was taken into account in the numerical analysis of the inhibitory assay experiments (see below) The Khyd value is directly related to the difference in free-energy between the virgin and modified inhibitors in solution This might be due either to a higher freeenergy content of virgin LCTI, corresponding to a conformational strain within the inhibitory loop, or to a particularly low level in free-energy of its modified form As will be discussed later, we did not find evidence for any notable deviation of the inhibitory domain geometry, or of the overall LCTI structure, in comparison to other available structures of BBIs, which could account for a particular conformational energy strain The gain in free-energy originates from a significant difference in the kon and k*on parameters (1.1 · 106 m)1Ỉs)1 vs 0.002 · 106 m)1Ỉs)1), as the corresponding koff and k*off values are very similar It is worth mentioning that the measured rate constants are consistent to those found previously [40] for soybean trypsin inhibitor, with the only exception being k*on The solution structure of LCTI is equivalent to the other reported BBIs [31,32] The overall molecular structure consists of two repetitive antipodal doublestrand b-sheets, each enclosing a type-VIb loop and bearing two distinct inhibitory sites The presence of a pseudo-dyad axis is also reflected by the very similar patterns of the C.S.I values measured for the two inhibitory domains, at the level of the 11–25 and 37–51 segments Relevant local differences in the amino acid sequence not seem to significantly alter the global structure, due to the strong cross-linking role of the disulphide bonds The generally conserved tertiary structure and hydrogen bond network give reason to the observed high thermal stability over a wide range of pH values and makes the inhibitor suitable for optimal binding with trypsin Other residues, not directly involved in the trypsin surface and catalytic FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS 4033 Inhibitory properties and NMR structure of a lentil BBI E M Ragg et al pocket recognition, should therefore be responsible for the high Khyd value A few amino acids, localized in the nonconserved regions of BBI trypsin inhibitory domain, are peculiar to LCTI and to all other proteins belonging to the same gene class [8] and might be connected to this unusual feature In synthetic trypsin inhibitor peptides [42,43], the nature of the residue at position P2¢ has been noted to modulate the rate of hydrolysis In particular, an increase in the hydrophilic character, such as in a Ile-Gln substitution, would favour peptide hydrolysis Indeed, as pointed out by Sonnante et al [8], LCTI has Gln18 at position P2¢ This substitution does not seem, however, to be detrimental to the association with trypsin, because a Kd value actually falling into the nanomolar range has been measured, in contrast to the finding of short peptides [43] The sequence ‘TRSQ’, corresponding to the P2-P2¢ region of LCTI, is quite uncommon within the Bowman–Birk family, it only being present only in Lens culinaris [8], in the highly homologous Vicia angustifolia inhibitor and in one isoform of sBBI [44] The measured lower value of k*on compared to kon might be related to an increase in conformational mobility, rendering the hydrolysed form less suitable to rapidly interact with trypsin and leading to inefficient resynthesis of the peptide bond In Bowman–Birk inhibitors, the native b-hairpin conformation is expected to be stabilized by a hydrogen bond network involving the side chains of residues P2, P1¢ and P5¢, which help to maintain the optimal conformation also in the hydrolysed form [44] The importance of Ser, a highly conserved residue at P1¢ position, has however, been questioned [45] The synthetic 11-residue cyclic peptide, corresponding to the core reactive site loops of both Bowman–Birk inhibitor and sunflower trypsin inhibitor (SFTI) proteins, represents at the moment, the shortest peptide with a canonical scaffold, endowed with high inhibitory activity [46] Addition of other residues at the N- and C-terminus, however, helps in increasing proteolytic stability, proving the critical role of distant amino acids in fixing the conformation of the hydrolysed form [42] A certain degree of dynamics in the BBI structure has been reported to be retained upon complex formation [47] The effect of single amino acid replacement on the hydrolytic stability was studied in detail for the ovomucoid third domain at pH [38] In that case, a small effect of P37¢ substitution was observed and explained in terms of entropic effects, due to interactions of such residue with the amino acid at position P2¢ The crystal structure of the unbound form of the tomato inhibitorII (TI-II) also revealed a significant conformational flexibility in the reactive site loop [48] Here, two pairs 4034 of /,w torsional angles were measured at the level of the scissile bond (P1: )80°,0° and )60°,120°; P1¢: )60°,150° and )160°,166°), values very similar to what was measured in LCTI (Table 2) In the case of Cucurbita maxima trypsin inhibitor CMTI-V, the decrease in the inhibitory activity upon hydrolysis with trypsin and the human blood coagulation factor XII was studied in detail It was concluded that hydrolysis did not involve a major variation in secondary structure, but was rather favoured by an increase in entropy due to greater conformational mobility of the binding loop first fragment [49] Residues at positions P6¢ and P8¢ would particularly contribute to the proteolytic stability This should apply also to LCTI To this respect, another region important for defining the conformational properties of LCTI* is the 53–55 segment, found to only interact weakly with the antitrypsin domain because of a greater intrinsic mobility Within this segment, a low-field shift for the amide proton resonances is actually observed upon hydrolysis, suggesting a closer interaction with the P6¢-P8¢ segment in the antitrypsin b-sheet domain Thus, the amino acid at position P37¢ (His54 in LCTI) plays a pivotal role, where a rather bulky and mobile side chain would render difficult a close packing of neighbour segments in the native form Besides, His54 is located in a region with the highest positive electrostatic potential, generated by neighbour charged residues This might reflect into a higher mobility around the P1-P1¢ bond Actually, Thr15 has an increased solvent accessibility, with respect to the dyad-related Ala41 In conclusion, the unusual propensity of LCTI towards hydrolysis, observed also by NMR and MS spectrometry at acidic pH in the absence of trypsin, might be the result of a concomitant series of factors They include the nature of the amino acid at position P2¢ (Gln instead of Ile) and an increased conformational mobility of the segment 53–55, whose major role is to anchor the antitrypsin b-hairpin domain into its native conformation by means of an extensive hydrogen bond ⁄ ion pair network Given the suggested defensive role of BBIs in leguminous seeds against insects, this inherent conformational mobility might provide a mechanism by which the inhibitor can balance the need for tight binding with the need for broad inhibitory function [48] Experimental procedures Extraction and purification of BBI from Lens culinaris seeds Dehulled lentil seeds (Lens culinaris, L., var Macrosperma) of commercial origin (600 g) were ground to a meal The FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS E M Ragg et al resulting flour was sieved through a 60 mesh metal sieve and suspended (1 ⁄ 10, w ⁄ v) in 100 mm sodium acetate buffer, pH 4.5, for h at °C under mild stirring The suspension was then sonicated (15 microns of amplitude) on ice, five times for 30 s every 15 s, using a Soniprep150 MSE apparatus (Fison, Crawley, UK) and centrifuged at 10 000 g at °C for 20 (J2-21M/E, Beckmann Instrument, Palo Alto, CA, USA) The proteins contained in the supernatant were precipitated with ammonium sulphate (70% saturation) and centrifuged as described above The pellet was dissolved with distilled water and dialysed at °C against Milli-Q water overnight The solution was heated in a water bath at 80 °C for 10 min, cooled on ice and centrifuged as already described The clear supernatant was brought to pH 3.0 with 0.2 m HCl in drops and then to pH 4.5 with 100 mm Tris ⁄ HCl buffer, pH 8.0 After centrifugation, the buffer of the supernatant was exchanged to 50 mm sodium acetate buffer, pH 4.5, by using an ultrafiltration apparatus (cut-off 3000 Da; Amicon, Bedford, MA, USA) and the crude extract was kept frozen at )20 °C until use About 20 mL of crude extract were loaded to a DEAE-cellulose column (2.2 · 130 cm, Whatman, Maidstone, UK) equilibrated with 50 mm Tris ⁄ HCl buffer, pH 8.0 The elution of the retained proteins was carried out stepwise with the same buffer containing 0.2, 0.4 and 0.5 m NaCl The fraction eluted with 0.2 m NaCl displayed the highest trypsin and chymotrypsin inhibitory activity The unretained fraction, which also displayed inhibitory activity, was neglected in this study After desalting by gel filtration, the active fraction was loaded onto an HPLC MonoQ column (0.5 · cm, Amersham Biosciences, Milano, Italy) equilibrated with 50 mm Tris ⁄ HCl buffer, pH 8.0 The retained proteins were eluted with linear gradient from to 0.3 m NaCl in 30 Of the seven fractions obtained, only the third, eluted with the buffer containing 0.15 m NaCl showed inhibitory activity This latter fraction was submitted to a trypsin-agarose affinity chromatography (TAC, Sigma-Aldrich, Milano, Italy) The column (1 · 2.5 cm) was equilibrated with 20 mm Tris ⁄ HCl buffer, pH 7.2 and the bound proteins were eluted by using a mm HCl solution Final yield was 30 mg of purified protein The homogenous protein is referred to as Lens culinaris trypsin inhibitor Mass spectrometry and amino acid sequencing Matrix-assisted laser desorption ionization ⁄ time of flight (MALDI-TOF) mass spectrometric analyses were performed by using a Bruker Daltonics Reflex IV instrument (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser (337 nm) and operated in linear mode with a matrix of sinapinic acid in 0.1% trifluoroacetic acid ⁄ CH3CN, : External standards, ranging from to 16 kDa (Bruker protein calibration standard) were used for calibration Inhibitory properties and NMR structure of a lentil BBI Amino acid sequence analyses were performed using a pulse liquid sequencer (Procise 491, Applied Biosystems, Foster City, CA, USA) following reduction and carbamidomethylation of the protein LCTI (0.2 mg) was dissolved in m urea, 50 mm dithiothreitol, 100 mm Tris ⁄ HCl, pH 8.6 The mixture was deoxygenated under vacuum and incubated overnight at 37 °C The reduced peptide was treated with iodoacetamide (0.1 mL of a 0.625 m solution in 100 mm Tris ⁄ HCl, pH 8.6) in the dark for 45 The carbamidomethylated LCTI was purified from the reaction mixture on a HPLC mod 510, equipped with a detector 2487 and SymmetryÒ C18 column (Waters, Milano, Italy) The two buffer system consisted of 0.1% trifluoroacetic acid in Milli-Q water (buffer A) and the same buffer containing 80% acetonitrile (buffer B) After elution with buffer A for at a flow rate of 0.8 mLỈmin)1, a gradient to 75% of buffer B in 75 was applied An aliquot of the material (200 pmol) was used to determine the N-terminal sequence of the entire polypeptide, allowing the identification of the first 30 residues The remaining part was digested with sequence grade Lys-C at a molar [E] ⁄ [S] ratio of : 350 in 25 mm Tris ⁄ HCl buffer, mm EDTA, pH 8.5 at 37 °C for 18 h The peptides were separated on a SymmetryÒ C18 column under the same conditions described above The recovered peptides were vacuum-dried and submitted to amino acid sequencing Alignment of the peptides was based on the N-terminal sequences of the entire protein, of a fragment obtained following incubation with trypsin (as detailed below) and of homologous BBIs (Fig 2) Antitryptic and antichymotryptic inhibition assays Trypsin (TPCK-treated from bovine pancreas), a-chymotrypsin (TLCK-treated from bovine pancreas), BApNA and GPpNA were purchased from Sigma-Aldrich Solutions of BApNA and GPpNA were freshly prepared by dissolving suitable amounts of the chromogenic substrate in doubledistilled water, 150 mm Tris ⁄ HCl, mm CaCl2, pH 8.2 Concentrations were checked by absorbance measurements on an aliquot of substrate solution after complete enzymecatalysed hydrolysis (p-nitroaniline: k ¼ 410 nm, e ¼ 8800 m)1Ỉcm)1) The reaction solutions contained BApNA and GPpNA at concentrations between 100 lm and 300 lm in 75 mm Tris ⁄ HCl, mm CaCl2, pH 8.2 Enzymes and LCTI were dissolved in the same buffer, at concentrations varying between 0.05 lm and 0.5 lm and between 0.01 lm and 0.5 lm for enzymes and inhibitor, respectively Inhibitor concentrations were checked by UV absorbance at 280 nm using a molar extinction coefficient value calculated on the basis of the amino acid sequence (e ẳ 4680 m)1ặcm)1) In the antitrypsin and antichymotrypsin activity assays, hydrolysis of the chromogenic substrates was continuously monitored at 410 nm, sampling the absorbance every 15 s for a FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS 4035 Inhibitory properties and NMR structure of a lentil BBI E M Ragg et al 40 time span, a few seconds after reagent mixing The UV-visible spectrophotometer was a PerkinElmer Lambda25 (Milano, Italy), equipped with a thermostatted cell Temperature was set at 37 °C In order to ascertain the exact point of trypsin-catalysed cleavage, LCTI (0.6 mg) was incubated for 0–6 h with 2.2 mL of trypsin 0.5 lm at 37 °C (100 mm Tris ⁄ HCl, 10 mm CaCl2, pH 8.2) Aliquots containing 0.15 mg of LCTI-LCTI* mixture were dialysed (cut-off 3500 Da) and, after lyophylization, reduced and carbamidomethylated The fragments were separated on SymmetryÒ C18 column Under these conditions, only peaks corresponding to the intact protein and fragments derived from cleavage at P1-P1¢ were detected, as proved by amino acid sequence analysis Numerical analysis of the inhibitory activity The absorbance data sampled during the tryptic and chymotryptic inhibition assays were converted into product concentrations and analysed using the scilab (v 2.0) software package (Copyright ª 1989–2005 INRIA ENPC, Paris Cedex 05, France) On the basis of the assumed kinetic model [25], a script was devised in order to solve the following system of ordinary differential equations by numerical methods: > dẵP ẳ dẵS ẳ kcat ẵE ẵS > > dt > dt KM > > > > dẵI > > < ẳ koff ẵC kon  ½EŠ  ½IŠ dt > d½CŠ > > kon ẵE ẵI koff ỵ k ị ẵC ỵ k ẵE ẵI > off on > dt ẳ > > > > dẵI > : ẳ k ẵC k E ẵI off on dt 1ị where [S], [P], [I] and [I*] are the actual concentrations of the substrate (BApNA or GPpNA), their hydrolysis products, and the inhibitor (respectively in the virgin and modified form); [C] is the concentration of the enzyme-inhibitor (virgin or modified) complex; kcat and KM are the kinetic and Michaelis–Menten constants relative to hydrolysis of the chromogenic substrates The first member of Eqn (1) assumes steady-state conditions and is valid for KM >> [S] Fitting the experimental data in the absence of inhibitor with the same script derived the kcat ⁄ KM ratio, as it was sufficient to set the initial LCTI concentration to zero Values for kcat were independently found equal to 135 min)1 and to 3.85 min)1 and KM values of 1250 lm and 850 lm for BApNA and GPpNA, respectively, by means of standard Lineweaver–Burk analysis of initial rate kinetics Fitting of the experimental data required as input data the initial concentrations of BApNA, enzyme and inhibitor, both in its virgin (I) and modified (I*) forms The initial amount of I* was estimated (10%) by 1H-NMR and RP-HPLC 4036 H-NMR spectroscopy LCTI was dissolved in 0.6 mL D2O (99.9% isotopic purity, ISOTEC, Miamisburg, OH, USA) or in H2O ⁄ D2O (90 : 10 v ⁄ v) mixture, at concentrations between 0.1 and mm No buffer or salt was added pH was adjusted to 3.1 (uncorrected pH values for deuterium effect), by addition of diluted HCl Solutions were immediately transferred into mm O.D NMR tubes (Wilmad, Buena, NJ, USA) NMR spectra were performed at temperatures ranging between °C and 40 °C on an AMX-600 spectrometer (Bruker Spectrospin, Rheinstetten, Germany), equipped with a mm inverse probe and z-axis gradients Spectra were referenced on external DSS, set at p.p.m Two-dimensional homonuclear correlation spectra NOESY [50], COSY-dQF [51] and TOCSY [52] were acquired using standard pulse sequences in phase-sensitive mode Typically, 800 · 2048 spectra were acquired using time proportional phase increments [53] and transformed to a final 2K · 2K real data matrix after apodization with a 90° and 60°-shifted sine-bell squared function in f2- and f1domain, respectively Baseline correction was achieved by a 5th-degree polynomial function TOCSY spectra were performed at various temperatures with a spin-lock value set at 0.045 s Solvent suppression was achieved either by presaturation and NOESY-type pulse sequences [50], or by gradient-based pulse sequences [54] in the case of D2O and H2O ⁄ D2O solutions, respectively For the quantitative evaluation of NOE interactions, a set of three consecutive experiments with tmix ¼ 0.08 s, 0.12 s and 0.35 s was performed at 25 °C Data processing was performed using xwinnmr software (v 2.6, Bruker Spectrospin) on a Silicon Graphics (Mountain View, CA, USA) INDY workstation 2D-spectral analysis and cross-peak integration were performed with sparky [55] 2D-cross-peak intensities were translated into NOE-distances by applying the two-spin approximation dij ¼ dref · (aij ⁄ aref)1 ⁄ using as reference the tyrosine proton pairs situated in ortho position (dref ¼ ˚ ˚ 2.40 A) as well as geminal protons (dref ¼ 1.80 A) Distance ˚ and +1.0 A The NOE-distances ˚ errors were set as )0.5 A were used only in the initial stages of the restrained simulated annealing procedures Final refinements were achieved by directly using the NOESY cross-peaks intensities measured at all mixing times and a complete relaxation matrix approach (see below) The / angles torsional restraints were calculated from measured 3JNa coupling constant values The / angles were restrained to )139 ± 30° for JNa > Hz and to )60 ± 30° for JNa < Hz Other / angles relative to residues detected in b-sheet secondary structure elements were restrained to )139 ± 30° in the early stages of structural refinement Chemical shift temperature coefficients for amide protons were measured from TOCSY experiments performed between °C and 35 °C Solvent exchange rates were classified according to the observed persistence of the amide NMR FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS E M Ragg et al signal in D2O solution at 25 °C: very slow exchange: kex < · 10)3 min)1; slow exchange: · 10)3 < kex < 40 · 10)3 min)1; medium exchange: 40 · 10)3 < kex < 600 · 10)3 min)1; fast exchange: kex > 600 · 10)3 min)1 Solution structure calculation An initial randomly folded polypeptide chain and its topology file were generated by xplor [56] Topologies and charges were taken from the ‘topallhdg.pro’ file present in the xplor library Torsional parameters for Pro20 and Pro46 were defined by setting the appropriate patch for cisprolines At this stage, no disulphide bond was defined A simulated annealing (SA) protocol was then applied [57] in order to create an initial set of 150 structures During simulation, the folding of the polypeptide chain was driven by / torsional angle constraints and a reduced set of longrange NOE distances, taken from the NOESY experiment performed at tmix ¼ 0.08 s These structures were subjected to a further SA, using all the NOE-derived interproton distances measured at tmix ¼ 0.08 s and all / torsional angle restraints Other distance restraints included hydrogen bonds for the slowly exchanging amide protons, residing in the b-sheet regions and reported in Fig In order to introduce proper directionality, for each observed hydrogen bond a pair of distance restraints was actually defined, namely ˚ ˚ d(C) ¼ O,H(N) ¼ 2.0 A and d(C) ¼ O,N(H) ¼ 3.0 A [23] From the initial set, the 55 best structures were extracted on the basis of NOE-restraints violations and were subjected to a second energy refinement step with the full relaxation matrix approach [58], where the NOE-based distances were substituted with all NOE cross-peak volumes measured at three mixing times A correlation time optimum value of ± ns was found by systematic search, assuming isotropic motions A further selection of the best 24 structures was made on the basis of total energy Finally, the ionization state for all ionisable side chains was defined for pH 3.1 Force-field was switched to Charmm22 [59], 11 chloride atoms were added and the protein was soaked with a solvation layer consisting of 600 water molecules In order to limit electrostatic interactions, the charges of the N-terminus and of Lys, Arg and His side chains were reduced to 0.2 units [60] In order to achieve convergence and quality for the final set of structures, all structures were subjected to a final refinement with the full relaxation matrix approach, consisting of several steps of restrained Molecular Dynamics (MD) at low temperature (20K)100K) followed by 300 steps of restrained energy-minimization During this final stage of refinement, torsional angle restraints were replaced by vicinal coupling constant restraints Chirality was checked during all SA steps with whatif [61] Final quality control was performed with procheck v 3.4.4 [30] Electrostatic potential calculations, molecular graphics and rendering were made with the aid of vmd v 1.8 [62] The best 20 structures, selected on the basis of lowest total energy, NMR R-factors and Ramachandran Inhibitory properties and NMR structure of a lentil BBI plot analysis, have been deposited in the Brookhaven Protein Data Bank (PDB code: 2AIH), together with the restraints used for the structure generation Chemical shift values have been deposited in Biological Magnetic Resonance Data Bank (BMRB code: 7078) NMR R-factors were calculated as R ¼ S[(Iic )1 ⁄ ) (Ii°)1 ⁄ 6] ⁄ S(Ii°)1 ⁄ 6, where Ii° and Iic are the normalized observed and calculated NOESY cross-peak intensities, respectively [56] Local rmsd values were calculated with data smoothing after superimposition of the deposited structures with a five-residue window Acknowledgements This work was supported by two grants from MIUR (Project FIRST-2004) References Bowman DE (1944) Fractions derived from soybeans and navy beans, which retard tryptic digestion of casein Proc Soc Exp Biol Medical 57, 139–140 Birk Y, Gertler A & Khalef S (1963) A pure trypsin inhibitor from Soya beans Biochem J 87, 281–284 Birk Y (1985) The Bowman-Birk inhibitor: trypsin and chymotrypsin inhibitor from soybean Int J Pept Prot Res 25, 113–131 Deshimaru M, Yoshimi S, Shioi S & Terada S (2004) Multigene family for Bowman-Birk type proteinase 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Determination of the aggregation state of LCTI by size exclusion chromatography Fig S6 Determination of the aggregation state of LCTI by DOSY Fig S7 Ramachandran plot and statistics of the 20 deposited structures Fig S8 Ensemble Ramachandran Plot for the 20 deposited structures Table S1 Experimental vicinal coupling constants and calculated values for the most representative structure This material is available as part of the online article from http://www.blackwell-synergy.com FEBS Journal 273 (2006) 4024–4039 ª 2006 The Authors Journal compilation ª 2006 FEBS 4039 ... the basis of lowest total energy, NMR R-factors and Ramachandran Inhibitory properties and NMR structure of a lentil BBI plot analysis, have been deposited in the Brookhaven Protein Data Bank... equal to 135 min)1 and to 3.85 min)1 and KM values of 1250 lm and 850 lm for BApNA and GPpNA, respectively, by means of standard Lineweaver–Burk analysis of initial rate kinetics Fitting of the... trifluoroacetic acid ⁄ CH3CN, : External standards, ranging from to 16 kDa (Bruker protein calibration standard) were used for calibration Inhibitory properties and NMR structure of a lentil BBI Amino