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 Keywords Bowman–Birk inhibito
Trang 1Bowman–Birk protease inhibitor from lentil
(Lens culinaris, L) seeds
Enzio M Ragg1, Valerio Galbusera1, Alessio Scarafoni1, Armando Negri2, Gabriella Tedeschi2, Alessandro Consonni1, Fabio Sessa1and Marcello Duranti1
1 Department of Agri-Food Molecular Sciences, Universita` degli Studi, Milano, Italy
2 Department of Animal Pathology, Hygiene and Veterinary Public Health-Section of Biochemistry, Universita` degli Studi, Milano, Italy
Bowman–Birk inhibitor (BBI) proteins are serine
prote-ase inhibitors First isolated from soybean seeds by
Bowman [1] and subsequently characterized by Birk
et al [2], BBIs are found in several plant sources,
spe-cially 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
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 5 July 2006)
doi:10.1111/j.1742-4658.2006.05406.x
Bowman–Birk serine protease inhibitors are a family of small plant pro-teins, 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 tryp-sin⁄ 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)1Æs)1 vs 0.002 lm)1Æs)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 physiolo-gical pH, the final structure results in an asymmetric distribution of oppos-ite 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 analog-ous BBIs, thus rendering the protein susceptible to hydrolysis The inhibi-tory 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
Abbreviations
BApNA, N-benzoyl- DL -arginine-p-nitroanilide; BBI, Bowman–Birk inhibitor; COSY-DQf, two-dimensional correlation spectroscopy double-quantum filtered; C.S.I., chemical shift index; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt; GPpNA, N-glutaryl- L -phenylalanine-p-nitroanilide; 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.
Trang 2identified [7,8] Despite their pronounced
microhetero-geneity, BBIs share a relatively high degree of sequence
homology, especially in the inhibitory domains, and a
highly conserved disulphide bridge network [9],
form-ing a consensus motif (Prosite code: PDOC00253)
There have been various hypotheses on the
physiolo-gical function of BBIs, including defence and
protec-tion, developmentally regulatory and sulphur-storage
roles, with no conclusive definition as yet [10] Plant
cell biology data on BBIs biosynthesis and
transloca-tion to the secretory pathway are also missing
From the inhibitory viewpoint most BBIs, especially
those from dicotyledonous seeds, have a
double-headed structure bearing two independent proteinase
binding sites, often one trypsin and one chymotrypsin
domain Various synthetic peptides consisting of a
sin-gle inhibitory domain and bearing the inhibitory
activ-ity have been produced and this has served to identify
the role of specific amino acid residues in the
protein-ase inhibition [11]
The renewed interest for this class of protease
inhibi-tors [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
cardio-vascular diseases Furthermore, the search for novel
biologically active protein molecules and their
exploita-tion as drugs or nutraceutical agents imply their
func-tional and structural characterization Based on these
considerations, the identification of novel BBI
inhibi-tors, either as natural compounds or synthetic
pep-tides, and the elucidation of their structural and
functional properties, is extremely important A recent
review dealt with legume-derived inhibitors [21]
We present here our results on the isolation, func-tional and structural analysis of a BBI from lentil (Lens culinaris L var Macrosperma) seeds Our isola-tion procedure yielded a protein in sufficient amounts and purity to obtain the complete amino acid sequence and1H-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 calcula-tions 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 nat-ural Bowman–Birk inhibitors
Results
Purification, mass spectrometry analysis and primary structure determination of LCTI The purification of LCTI from lentil seeds involved var-ious chromatographic steps, including a final affinity chromatography step on agarose-immobilized trypsin The antitrypsin activity was measured at every purifica-tion 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-ter-minal amino acid sequencing, mass spectrometry (MALDI-TOF) (Fig 1), amino acid sequence analysis
of Lys-C generated fragments and1H-NMR The isola-ted 67 amino acid protein had the same primary struc-ture 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, inclu-ding 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 2 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 con-servative substitutions were observed at the inhibition
Trang 3sites, 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
chromo-genic substrates BApNA and
N-glutaryl-l-phenylalan-ine-p-nitroanilide (GPpNA) in the presence of bovine
trypsin and a-chymotrypsin, respectively, and
increas-ing amounts of LCTI Figure 3 reports the amount of
hydrolysed BApNA as a function of time In the
pres-ence of LCTI, two distinct kinetic regimes with
differ-ent rate constants were presdiffer-ent 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-ter-minal P1-P1¢ bond (not shown)
The kinetic model assumed (Scheme 1) implies the formation of a 1 : 1 complex [25] and is the simplest one able to fit with sufficient accuracy the experimental results
The kcat⁄ KMratio was derived by fitting the experi-mental 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-Fig 2 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.
Fig 3 Hydrolysis of 213 l M BApNa as function of time in the pres-ence of 0.1 l M trypsin (pH 8.2, 37 C) and the following amounts
of LCTI: 0 l M (¯, curve 0), 0.038 l M (*, curve 1), 0.11 l M (·, curve 2), 0.225 l M (h, curve 3).
Fig 1 MALDI-TOF mass spectrum of LCTI Sin: sinapinic acid The
insert shows an expansion of the molecular peak.
Scheme 1.
Trang 4lysed form (LCTI*), leading to an accurate
measure-ment of k*on and k*off At [LCTI]⁄ [trypsin] ¼ 1.1 and
2.25 the conversion of LCTI into LCTI* (Fig 3,
curves 2 and 3), allowed the simultaneous computation
of koff, k*on and k*off The konvalue was assumed by
analogy with soybean BBI [26] The two dissociation
constants (Kd¼ koff⁄ konand K*d¼ 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 1
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
import-antly, to a minor difference between Kd and K*d
(Table 1)
1H-NMR sequential assignments and secondary
structure determination
A total of 62 NH-Ha interactions were detected
through the analysis of the TOCSY and
two-dimen-sional correlation spectroscopy double-quantum
filtered (COSY-DQf) experiments, allowing the
identifi-cation 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
NH-CHa-CH2b 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
solu-tion As residue 16 is located in the antitrypsin site,
this form was attributed to LCTI* Additional
reso-nances, 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
corres-ponds to a mass increase of 18 Da, as expected from
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 inhib-itor was kept in solution at pH 3.1 for few days, suggesting a particular intrinsic lability of the Arg16-Ser17 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 4 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 1 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)6values taken from [26].
kon· 10)6( M )1Æs)1) k
off · 10 3 (s)1) k* · 10)3( M )1Æs)1) k*
off · 10 3 (s)1) Kd· 10 9 ( M ) K*d· 10 9 ( M ) Khyd
Fig 4 LCTI b-hairpin elements (segments Thr11-Val25 and Lys37-Tyr51), with observed NOE interactions (double-arrow) and hydro-gen bonds involving the slowly exchanging amide protons (dotted line) T and CT denote the antitrypsin and antichymotrypsin sites, respectively.
Trang 5together 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
exten-ded 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
reduc-tion in propensity for an extended conformareduc-tion
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 structure-less, 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 Deuter-ium 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 coef-ficients (DdNH⁄ DT) were determined by performing a series of TOCSY experiments at various temperatures (Table 2) As absolute values less than 5 p.p.b.ÆK)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 struc-ture, will be discussed below
Solution structure of LCTI The observed NOEs also provided information on the global protein folding All the measured vicinal coup-ling constants and NOE interactions were translated into restraints for the generation of the solution struc-ture Statistics for the total amount of experimental data are reported in Table 3
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 (tmix¼ 80 ms), was utilized for generating a starting restraints set, together with ideal values for /,w dihed-ral angles One hundred and fifty structures were thus obtained and analysed in terms of total energy,
Fig 5 (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.
Trang 6restraint 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
minimi-zation 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
switch-ing the force-field to Charmm22 Vicinal couplswitch-ing
con-stants 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
cor-relation time value of 7 ± 1 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 exclu-sion 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 6 reports the superim-posed Cachains 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 ele-ments, connected by segments with augmented conformational mobility The antitrypsin domain comprises the 11–25 segment, incorporating an anti-parallel 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-con-formation, as previously discussed The corresponding /,w-values are reported in Table 4 in comparison with those obtained from the X-ray structure of Pisum sati-vum trypsin inhibitor isoform IVb (PSTI-IVb) [31] Folding similarities of LCTI with PSTI-IVb and soy-bean Bowman–Birk inhibitor (sBBI) are shown in Fig 7, reporting the superimposition of Ca carbons (rmsd 1.99 and 2.10 A˚, respectively, calculated consid-ering 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 < 5 p.p.b.ÆK)1) and the very slow solvent exchange rates of the corres-ponding amide protons (kex< 3· 10)3min)1)
Thr21-NH 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 coef-ficients, are not fully exposed to solvent This indicates that these residues are involved in other tertiary inter-actions, in particular with the 52–55 segment A spatial
Table 2 Temperature coefficients (Dd ⁄ DT) and deuterium
exchange rates (k ex ) of LCTI amide protons Estimated accuracy of
temperature coefficient is ± 0.1 p.p.b.ÆK)1.
Residue
Dd ⁄ DT
(p.p.b.ÆK)1)
kex· 10 3 (min)1) Residue
Dd ⁄ DT (p.p.b.ÆK)1)
kex· 10 3 (min)1)
Trang 7interaction actually exists between Thr21 and Thr53 methyl groups and between Cys22 and the Thr53-Lys55 segment This latter segment is oriented perpen-dicular 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 coup-ling 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 (rmsd¼ 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,
Gln49-NH⁄ 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 pro-tons 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,
Arg28-NH forms a strong hydrogen bond with Cys48-CO (kex< 3· 10)3 min)1) The observed Arg28-NH⁄ Cys48-CO interaction is supported also by long range NOEs (Val27-Ha⁄ NH and Arg28-NH ⁄ Cys48-NH) 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 val-ues and temperature coefficients of the amide protons, suggesting a closer interaction with the antichymotryp-tic 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 pep-tide backbone folds into a sharp turn
Relevant hydrogen bonds were found between Asp10-NH⁄ Asp26-COOH and Asp36-NH⁄ Asp52-COOH 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
Table 3 Statistics for the 20 best structures derived from the
restrained MD calculations rmsd, root mean square deviation.
Conformational energy parameters E (kcalÆmol)1)
Deviations from average
‘‘topallhdg’’ structures
‘‘Charmm22’’ structures
Ramachandran plot statistics
Amino acids in generously allowed region 4.3%
Number of restraints
Restraint deviations
Distances
H-bond
Coupling constants
Restraint deviations (rmsd)
Trang 81H-NMR spectra have been assigned, i.e sBBI [32]
and MSTI [23] However, Asp36 is not a highly
con-served residue, because in MSTI and sBBI a lysine is
present at that position, whereas PSTI-IVb has a
leu-cine 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
part-ners, the highly conserved Asp26 and Asp52 residing,
respectively, at the end of the antitrypsin and
anti-chymotrypsin 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 pro-vides high thermal stability and restricted conforma-tional 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 poten-tial 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 distribu-tion of opposite charges, at pH values around neutral-ity (Fig 9) The electrostatic potential is unevenly distributed on the protein surface, as a negative poten-tial is calculated at the C-terminus, near the antichymo-tryptic site, whereas the antiantichymo-tryptic 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 dimeriza-tion, 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
Fig 6 Superimposition of the best 10 LCTI structures derived from restrained simulated annealing calculations Caatoms only are dis-played.
Table 4 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.
LCTI
PSTI-IVb
18.4 ± 9.2
)63.2 ± 0.2
Trang 9trypsin⁄ 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
dicoty-ledonous 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
previ-ously mentioned, lentil seeds contain various BBI
iso-forms 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
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, how-ever, 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 refer-ence 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 Bow-man–Birk inhibitor isolated from a natural source By contrast, the kinetic and thermodynamic parameters, derived for the antichymotryptic activity of LCTI,
Fig 8 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).
Fig 7 Superimposition of the LCTI solution structure (PDB ID 2AIH, red) with PSTI-IVb (PDB ID 1PBI, green) and sBBI (PDB ID 1BBI, blue).
C a atoms only are displayed.
Trang 10reside 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
depend-ence of such a parameter on pH [38]
For some inhibitors, complex formation is a
two-step 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
sta-ble 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
previ-ously identified BBI from Lens culinaris (LCI-1.7) [41],
a value likely to correspond to an LCTI⁄ LCTI*
mix-ture In our case, the initial presence of LCTI* was
taken into account in the numerical analysis of the
inhibitory assay experiments (see below)
The Khydvalue 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
free-energy 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 evi-dence 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· 106m)1Æs)1 vs 0.002· 106m)1Æs)1), as the cor-responding 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 double-strand 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 do 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 rea-son 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
Fig 9 Particle mesh Ewald electrostatic potential calculated for LCTI at pH 6 Isopotential curves are displayed at )60 kTÆe )1(red) and at +60 kTÆe)1units (blue).