From the
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia,
Lehrstuhl für Biopolymere, Universität Bayreuth, Universitätsstrasse 30, Bayreuth D-95447, Germany,
|| The Wistar Institute, Philadelphia, Pennsylvania 19104
Received for publication, December 20, 2002
, and in revised form, February 10, 2003.
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ABSTRACT |
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INTRODUCTION |
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The new generation of circular peptides/proteins discovered in the last few years differs from previously known cyclic peptides such as cyclosporins in that the latter are generally not direct gene products but are synthesized in bacteria by multifunctional enzymes and often contain non-conventional amino acids (5). By contrast, recently discovered circular miniproteins such as the plant cyclotides (6) are gene products that are post-translationally processed to cyclize their conventional peptide backbone (7). Although in vitro cyclization procedures are now being developed for the synthetic production of circular proteins, little is known about the mechanisms and driving force behind in vivo cyclization of naturally occurring cyclic proteins. This is in part because limited knowledge about the locality of the NH2- and COOH termini of putative linear precursors of cyclic proteins makes predictions about cyclization mechanisms difficult.
More than 50 naturally occurring backbone cyclized proteins have now been reported (1), with three of these being proteinase inhibitors: MCoTI-I and MCoTI-II from Momordica cochinchinensis (8, 9), and SFTI-11 from sunflower seeds (10). The Bowman-Birk (BB) inhibitor SFTI-1, shown in Fig. 1, is the focus of the current study and is the smallest known cyclic plant peptide. It is the most potent and only known cyclic BB inhibitor, having a Ki value for trypsin in the sub-nanomolar range (1014).
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Interestingly, the cyclic backbone of SFTI-1 does not appear to be essential for the high potency, because we recently showed that SFTI-1[1,14], an acyclic permutant of SFTI-1, with a break in the backbone chain between residues 1 and 14 (see Fig. 1), also has a very high affinity for trypsin (11). Although its Ki value is apparently about 20-fold weaker than the cyclic form, this difference is not highly significant due to the difficulties of accurately determining Ki values for such potent inhibitors (15). Zablotna et al. (14) also recently reported that there is essentially no difference between the inhibitory potencies of cyclic SFTI-1 and this particular acyclic derivative. Thus, it would appear that structural conservation of the reactive loop of SFTI-1[1,14], rather than the cyclic backbone, is essential for potency and that a slight disordering of the open termini in the non-reactive loop of SFTI-1[1,14] does not significantly reduce binding efficiency to trypsin. Although the role of cyclization is not certain, it has been suggested that the cyclic backbone of native SFTI-1 possibly evolved to protect it against degradation by proteases to allow SFTI-1 to function over an extended in vivo lifetime (11). Understanding the mechanism of cyclization for SFTI-1 may therefore have potential applications in the pharmaceutical industry and for the design of improved peptidic drugs.
Nothing is known so far about the cyclization mechanism for SFTI-1 in planta, and there are no data describing enzymes with the specificity necessary to perform this task. Here we describe an in vitro enzymatic mechanism for the cyclization of a linear precursor of SFTI-1, SFTI-1[6,5] (Fig. 1), that is open at the scissile bond, i.e. the bond that would normally be cleaved in homologous substrates. Although this mechanism, based on the reversible equilibrium between species containing an intact and a cleaved peptide bond, is generally applicable to many serine proteinase inhibitors, SFTI-1 is the first example of an inhibitor in which the peptide bond resynthesis results in a backbone cyclic peptide. The gene sequence of the putative precursor protein of SFTI-1 is unknown, and the demonstration of enzymatic cyclization of a linear analogue of SFTI-1 potentially provides insight into a theoretically possible in vivo precursor.
In the current study we have gained a structural insight into five conformers/isomers of this putative acyclic precursor and have determined full three-dimensional structures for two of the conformers, which we compare with the structure of SFTI-1. In addition, anti-trypsin activity assays were carried out for SFTI-1[6,5], and the products of a 1:1 reaction mixture of trypsin and SFTI-1[6,5] have been analyzed. The results have provided an insight into potential mechanisms of cyclization of SFTI-1 in vitro and in vivo.
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EXPERIMENTAL PROCEDURES |
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NMR SpectroscopySamples prepared for NMR spectroscopy contained 2 mM SFTI-1[6,5] in 20 mM sodium phosphate (H2O/D2O, 90/10%, v/v) at pH 4.5. One-dimensional 1H-NMR spectra recorded over the temperature range 280310 K revealed that the dispersion of peaks was optimal at 286 K. Homonuclear two-dimensional 1H spectra were recorded at 286 and 298 K on a Bruker DMX750 spectrometer. NOESY and TOCSY spectra used a WATERGATE scheme (16) for water suppression. The mixing times for the NOESY spectra were 200 and 300 ms, and the mixing time for the MLEV17 sequence (17) was 80 ms. Water suppression in the DQF-COSY experiment was achieved using selective low power irradiation of the water resonance during a relaxation delay of 1.3 s. 16,384 data points were collected for one-dimensional spectra, and 4,096 data points in the t2 and 512 increments in the t1 dimension were collected for two-dimensional experiments. NOESY, TOCSY, and E.COSY spectra were recorded in D2O at 286 K. Slowly exchanging amide protons were identified by recording a series of one-dimensional spectra and two-dimensional TOCSY spectra at 286 K over a period of 12 h on a sample immediately after dissolution in 99.996% D2O. A sequence-specific resonance assignment was performed by standard methods (18). Cis proline residues were identified by strong H
(i)/Pro-H
(i+1) NOE signals in the NOESY spectrum, whereas a trans conformation was evidenced by unambiguous H
(i)/Pro-H
(i+1) NOE signals and the absence of H
(i)/Pro-H
(i+1) connectivities (18). 3JNH
coupling constants were obtained from a high resolution one-dimensional spectrum and from line-shape analysis of the anti-phase cross-signal splitting in a high digital resolution DQF-COSY spectrum using a Lorentzian function for peak fitting. The
1-angles for side-chain rotamers were obtained from line-shape analysis of H
H
couplings in an E.COSY spectrum together with NOE intensity patterns between resolved HNH
protons. Secondary chemical shifts were obtained using published random coil values (19), and specific corrections for residues preceding a proline (19) were taken into account.
Structure CalculationsDistance constraints were derived from the intensities of cross-peak signals from NOESY spectra at 286 K recorded with a mixing time of 200 ms. The spectrum at 298 K was used to resolve ambiguities due to spectral overlap. Distance restraints were divided into three groups according to the relative NOESY cross-peak intensities: strong, upper distance limit of 2.7 Å; medium, upper distance limit of 3.5 Å; weak, upper distance limit of 5.0 Å. All structures were calculated using X-PLOR 3.851 (20) with a modified ab initio simulated annealing protocol (21). The structure calculation strategy was based on a three-stage simulated annealing protocol (22, 23). For details see the Supplementary Material. An iterative approach using several rounds of structure calculations with subsequent distance analysis was used to solve ambiguities in NOE cross-peak assignments. Dihedral angle constraints were introduced in the later rounds of structure calculations. Distance constraints for hydrogen bonds were generated based on deuterium exchange data and by examination of structure families calculated without hydrogen-bond constraints. The 20 structures with lowest overall energies from the final 50 structures were retained for further analysis. Structures were visualized using the program MOLMOL (24) and analyzed with PROCHECK-NMR (25).
Trypsin AssaysTrypsin assays were carried out at 303 K as described by Korsinczky et al. (11). The concentration of SFTI-1[6,5] was determined by amino acid analysis. SFTI-1[6,5] (0150 nM) and trypsin (42 nM) were preincubated together for3hat303K.The reactions were initiated by addition of substrate and monitored at 405 nm with readings taken every 2 min with shaking between readings using a Molecular Devices SpectraMax 250. All reactions were carried out in duplicate, and the corresponding Ki value was determined using a tight-binding equation (26).
Mass Spectroscopy/Monitoring of the Trypsin-dependent Interconversion of SFTI-1[6,5] to SFTI-1SFTI-1[6,5] and bovine -trypsin (Type XIII L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated, EC 3.4.21.4
[EC]
) were incubated together at 303 K in a 1:1 ratio (350 nM each), at pH 8.0, 50 mM Tris HCl, 25 mM CaCl2. 20-µl aliquots were taken in duplicate after 2 min and every 15 min for the first 2 h and every 30 min for the next 2 h and then every hour. The reaction was stopped by a sudden pH drop to <1 by immediate addition of 5 µl of trifluoroacetic acid to the aliquots with vigorous shaking. The samples were subjected to LC-MS analysis, using an electrospray Qq time of flight mass spectrometer (QSTAR Pulsar, Applied Biosystems, Foster City, CA) operated under positive ion detection conditions: declustering potential of 80 V, focusing potential of 250 V, declustering potential 2 of 10 V, and ion spray sprayer level of 5000 V. Extracted ion chromatograms for the doubly protonated SFTI-1[6,5] (m/z 766.3) and SFTI-1 (m/z 757.3) were constructed, and the ratio of the two peaks was calculated using both peak area and height. The integrity of the initial measurements was determined by re-measuring the amounts of SFTI-1 and SFTI-1[6,5] from the initial reaction aliquots 1 day after the initial readings. An unchanged result for the two peptides proved that trifluoroacetic acid addition did not degrade either SFTI-1 or SFTI-1[6,5] and did completely stop the trypsin reaction.
Isolation of SFTI-1 from Sunflower SeedsSunflower seeds (about 1 week prior to maturation) were ground with dry ice, and the mixture was defatted with chilled petroleum spirit (10). The mixture was filtered, and a water extraction was made from the dried solid phase. The water extract was divided in two portions, and to eliminate possible protease activity one portion was treated with trifluoroacetic acid to drop the pH to <1. After centrifugation for 10 min at 15,000 rpm the supernatants of both extracts were directly subjected to LC-MS analysis and yielded identical results.
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RESULTS |
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Species A, the major species of SFTI-1[6,5], exists as two conformers (A1 and A2) (Fig. 3) evidenced by two sets of spin systems for residues S6 to P9 that differ only in cis/trans isomerization of P8, with the trans conformation (A2) being more abundant. For species B all three Xaa-Pro bonds adopt a trans conformation as for A2, but the backbone chemical shifts are notably different from those of species A (Fig. 2). The most striking feature that distinguishes species B from all the other species is the presence of an iso-aspartate residue at position 14, as deduced from the clear absence of a H(i)HN(i+1) sequential NOE, together with strong H
(i)HN(i+1) NOEs. Furthermore, the high 3JH
HN coupling constant (>9 Hz) of residue 14 of species B correlates with an extended
-angle of 120 ± 40°. This angle is easier to fulfill if the non-binding loop is extended by the additional methylene group resulting in a more relaxed ring structure. Species C exhibits an F12/P13 cis orientation, and the four NH2-terminal amino acids (S6P9) adopt two different conformations: one with P8 and P9 both in the trans orientation (C1), and one with P8 in the trans and P9 in the cis orientation (C2) (Fig. 3). For the NH2-terminal segment of C2, only the two proline residues could be definitively assigned. Out of the three species, A exhibits backbone chemical shifts that most closely resemble the corresponding shifts of native cyclic SFTI-1 (Fig. 4), and A1 exhibits the same geometry for all three prolines as cyclic SFTI-1 (11) (Protein Data Bank code 1JBL
[PDB]
).
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Secondary Structure of SFTI-1[6,5] Conformers/IsomersA summary of the secondary chemical shifts from I10 to T4 for all three SFTI-1[6,5] species relative to those of cyclic SFTI-1 is given in Fig. 4. Consecutive downfield shifted H resonances, indicative of extended structure, were found for G1 to C3 of both cyclic SFTI-1 and the SFTI-1[6,5] species A, with another distinctive downfield shifted value for C11. Species C exhibits consecutive upfield shifted H
resonances for residues F12 to D14, indicating helical or turn-like structure. For species B, no indications of regular secondary structure were found. These findings were supported by an analysis of the 3JNH
coupling constants. For species A they were >8 Hz for C3, C11, and I10, representing residues in an extended structure. For species B F12 and iso-D14 had coupling constants >8 Hz, and for species C only C11 exhibited a coupling constant typical of an extended conformation. No coupling constants <6 Hz, which are typical of an
-helix, were detected for any of the three species. Slow exchanging amide protons were only detected for I10, F12, G1, and R2 of species A. Further evidence for a
-sheet like structure for species A was deduced by a strong H
H
NOE between the two cysteine residues. In summary, evidence for regular secondary structure could only be deduced for species A, indicating a small anti-parallel
-sheet.
Structure CalculationsStructure calculations were performed for the four conformers and one isomer of SFTI-1[6,5]. Input data and structure statistics are summarized in Table I. The structure calculations provided well-defined structures for the two conformers of species A with a backbone r.m.s.d. for the final 20 structures of 0.49 Å for A1 and 0.45 Å for A2 (protein data bank codes: A1, 1O8Z [PDB] ; A2, 1O8Y [PDB] ).
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Species A exhibits a hairpin structure that is similar for both conformations (A1/A2) and closely resembles the corresponding region of cyclic wild-type SFTI-1, i.e. a short double-stranded anti-parallel -sheet connected by the disulfide bridge and stabilized by hydrogen bonds (Fig. 5). The difference between the two conformations is the cis/trans isomerization of P8. The I7P8 bond shows a cis orientation in the minor conformer A1, essentially maintaining the binding loop orientation, even though the backbone is open between residues K5 and S6. In the major conformer A2, P8 is in a trans configuration, giving the binding loop an extended conformation (Fig. 5).
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The structure calculations for species B and the two conformations of species C did not result in convergent structures due to limited NOE information. However, it could be deduced, from chemical shift data and the absence of certain characteristic NOEs, that the small -sheet found for both conformations of species A is not present in species B and C.
Trypsin Catalyzed Cyclization of SFTI-1[6,5]Although trypsin is normally used to facilitate proteolysis, i.e. peptide bond cleavage, this equilibrium reaction is in principle reversible, and we were interested to see if presentation of an acyclic precursor species would result in cyclization. Therefore, a 1:1 mixture of SFTI-1[6,5] with bovine -trypsin was incubated at 303 K (pH 8). Aliquots of the reaction mixture were taken over a 24-h period, and LC-MS analysis confirmed that cyclic SFTI-1 was indeed produced. The ratio of the intact and cleaved form of SFTI-1 was determined by quantitative analysis of the peaks with masses corresponding to SFTI-1[6,5] and cyclic SFTI-1 (Fig. 6). After 2 min of reaction time, cyclic SFTI-1 could be detected. A steady state was reached after
130 min and resulted in
90% SFTI-1 to 10% SFTI-1[6,5] (Fig. 6). Coelution experiments with added synthetic SFTI-1 and SFTI-1[6,5] confirmed that the resulting molecules are indeed the cleaved and intact inhibitor.
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The above procedure was repeated starting with cyclic SFTI-1 under the same conditions. After a reaction time of 2 h, cleaved SFTI-1[6,5] in a yield of about 7% was recovered. Therefore, a ratio of cyclic SFTI-1 to SFTI-1[6,5] of
9:1 was reached regardless of whether the reaction was initiated with SFTI-1 or SFTI-1[6,5].
Isolation and Verification of the Cyclic Nature of SFTI-1 in Sunflower SeedsThe previously reported isolation procedure of SFTI-1 from sunflower seeds (10, 27) involved the incubation of SFTI-1 on a trypsin affinity column for up to 2 h. Given that SFTI-1[6,5] can be 90% converted into the cyclic form when incubated with trypsin in a similar time frame and that Luckett et al. (10) reported a ratio of 9:1 of intact to cleaved SFTI-1 after trypsin affinity column purification, the question was raised as to whether the peptide is actually acyclic in its natural state and whether the exposure to trypsin on the affinity column may have made it cyclic. We therefore repeated the isolation of SFTI-1 from fresh sunflower seeds with a method similar to that described by Luckett et al. (10) but omitted the trypsin affinity column step. In the water extract the mass of the doubly charged ion of cyclic SFTI-1 (M++ = 757.32) was detected, with neither the single nor doubly charged ion of acyclic SFTI-1[6,5] being present. In a separate experiment, we added synthetic cyclic SFTI-1 to the water extract, and subsequent LC-MS analysis of this mixture resulted in one sharp peak with the correct mass for SFTI-1. This confirms unambiguously that SFTI-1 is present naturally in cyclic form in the seed.
Trypsin Inhibition AssaysTrypsin assays were carried out as previously described (11). SFTI-1[6,5] inhibited bovine -trypsin with a Ki of 0.54 ± 0.1 nM (Fig. 7), a result identical to that for cyclic SFTI-1. Interestingly though, cyclic SFTI-1[6,5] shows inhibitory activity with minimal incubation, whereas SFTI-1[6,5] requires a 2- to 3-h preincubation with trypsin to show substantial inhibitory activity, thus making SFTI-1[6,5] a less effective inhibitor. This delay in complex formation is further examined in the discussion section.
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DISCUSSION |
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The major species present in solution (A) exhibits a -hairpin structure from C11 to C3 similar to wild-type cyclic SFTI-1 and forms two conformers (A1 and A2) that differ only by a cis/trans isomerization about the I7/P8 peptide bond. The three-dimensional structure of the cis conformer A1 of SFTI-1[6,5] closely resembles the structure of cyclic SFTI-1. The C
atom r.m.s.d. value between the solution structures of A1 and SFTI-1 (1JBL
[PDB]
) is 0.98 Å and that between A1 and the crystal structure of SFTI-1 in complex with trypsin (1SFI
[PDB]
) is 1.21 Å, indicating that, although the backbone is open between residues K5 and S6, the binding loop orientation is essentially maintained (Fig. 5).
The second most abundant species (B) features backbone chemical shifts that differ notably from those of species A and also from those of cyclic SFTI-1. In this species all three Xaa-Pro peptide bonds are in a trans orientation and there is an iso-aspartate residue at position 14. The number of structural restraints for species B, however, was not sufficient to result in a family of converging structures, same as for species C.
Trypsin-catalyzed Cyclization of SFTI-1[6,5]In addition to defining the conformational properties of SFTI-1[6,5] we examined its reactivity. We have shown that the proteolytic enzyme trypsin is able to resynthesize the peptide bond between the P1/P1' residues (nomenclature according to Ref. 28) and consequently cyclizes the linear precursor SFTI-1[6,5] to form native cyclic SFTI-1. The cyclization reaction occurs very efficiently: a stable ratio of more than 9:1 of cyclic SFTI-1 to acyclic SFTI-1[6,5] is reached within 2 h. Resynthesis of a peptide bond between P1/P1' residues upon interaction with a suitable proteinase has previously been demonstrated for various proteinase inhibitors (29, 30), but the resynthesis of this bond in SFTI-1[6,5] is unique because it leads to a cyclic peptide chain rather than ligation of two chains, as is the case for other classic inhibitors. No matter which form of the inhibitor was incubated with enzyme, after a sufficient reaction time over 90% of cyclic inhibitor was present. This result proves that cyclic SFTI-1 and the cleavage product SFTI-1[6,5] are interconvertible and that the enzyme-inhibitor complex C is the same, no matter whether it was made from the proteinase E and the virgin inhibitor I or E, and the modified inhibitor I* (29), in accordance to the equilibrium nature of the canonical mechanism of inhibition given by Finkenstadt and Laskowski (8),
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From the crystal structure of SFTI-1 in complex with trypsin it is known that the peptide bond between K5 and S6 is intact in the inhibitory complex (10). Our experimental results show that this inhibitory complex can also be formed using SFTI-1[6,5] instead of SFTI-1, in accordance with Reaction 1. Consequently, both peptides have identical inhibition constants, and SFTI-1[6,5] itself is a strong trypsin inhibitor. However, complex formation using SFTI-1[6,5] is significantly slower than complex formation using cyclic SFTI-1, mostly because peptide bond formation has to occur as a preceding step when complex formation starts with SFTI-1[6,5], in accordance with an extended version of Reaction 1,
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In the case of SFTI-1[6,5] as I* an even more complex equilibrium is present, contributing further to its slower association rate, because it is not a structurally homogeneous compound but a mixture of conformers/isomers that are in mutual equilibrium. A consideration of geometric factors suggests that the orientation of the S6 to I10 binding loop arm, in particular the cis conformation of P8 in the P3' position, is a prerequisite for an efficient interaction of SFTI-1[6,5] with trypsin. Because our studies show that none of the multiple SFTI-1[6,5] conformers/isomers are present at more than 40% in solution, there must be equilibration between the isomers/conformers to produce >90% of cyclic SFTI-1 after reaction with trypsin, namely cis/trans isomerization of the proline residues. There has to be equilibration between the iso-D14 and D14 isomers as well, because about 30% of all molecules contain the iso-aspartate residue. It appears that, at the beginning of the incubation with trypsin, only a fraction of all SFTI-1[6,5] molecules present in solution, those adopting the A1 conformation, fulfills the requirements to fit into the active site pocket of the proteinase.
Mimicry of Larger Bowman-Birk InhibitorsLike all canonical serine proteinase inhibitors, proteins of the BB inhibitor family interact with the enzymes they inhibit via an exposed surface loop that adopts a canonical proteinase inhibitory conformation. A particular feature of full-length BB inhibitor proteins is that the interacting loop is stabilized by a well-defined disulfide-linked -sheet region. For small nonapeptides comprising the 9-amino acid active site loop and the disulfide bridge, this
-sheet is shortened and the reduced stabilization is accompanied by poorer inhibitory activities (32). These small synthetic peptide mimics of BB inhibitor proteins are able to bind to and inhibit target proteinases, but when cleaved at the active site peptide bond, they are incapable of being resynthesized by the proteinase (32, 33). NMR studies on such nonapeptides did not result in stable structures (34). In contrast to these small peptides, the
-sheet structure is intact in SFTI-1 and in both species A conformers of SFTI-1[6,5], even if the active site peptide bond is hydrolyzed. This stabilization is sufficient to make SFTI-1[6,5] amenable to resynthesis of the active site peptide bond. Thus, the intact
-sheet structure seems to be another prerequisite for the pre-orientation of the two "arms" of the cleaved inhibitory loop for an efficient interaction with the proteinase. The importance of flanking residues for the short
-sheet is also supported by the fact that a synthetic undecapeptide derived from a combinatorial peptide library screening also exhibits a very stable
-hairpin structure across the active site loop (35). One goal of studies on BB inhibitors has been a determination of the minimal requirements necessary to retain the proteinase inhibitory activity of full-length inhibitors (32, 33). All investigations so far have kept the canonical loop intact, and variations have been made in specific side chains. Here we report that it is not necessary to keep the P1/P1' site intact to maintain activity, as long as the scaffold of the loop, stabilized by the
-hairpin loop located opposite to the binding loop is maintained.
In Vivo Synthesis of Cyclic SFTI-1Little is known about the in vivo cyclization mechanisms of naturally occurring cyclic peptides/proteins, and it is likely that different cyclization mechanisms might apply to different peptides. Here we have shown that the cyclic nature of SFTI-1 is not an artifact of the previously used purification scheme (10) and that SFTI-1 occurs naturally in the cyclic form in the seed. The possibility that a small linear precursor (e.g. SFTI-1[6,5]) is cyclized via a reaction with native proteinases present in the seed constitutes one hypothesis for the in vivo synthesis of cyclic SFTI-1. Various serine proteinases that might be suitable for this task are indeed present in plant seeds (3640). However, the gene encoding SFTI-1 is not known and, hence, nor is the location of the native termini of a linear precursor peptide. Analysis of the genes encoding the cyclic peptide RTD-1 from Macaca mulatta has shown that more complicated cyclization mechanisms are conceivable and that the precursor is not necessarily one continuous peptide chain (41).
A second hypothesis for the synthesis of cyclic SFTI-1 can be derived from the fact that all known cyclic mini-proteins from plants appear to be derived from longer precursor proteins (1), and, therefore, both cleavage and cyclization steps seem to be involved in their synthesis. The majority of BB inhibitors from dicot plants comprise 6090 amino acid residues and have a symmetrical double-headed structure with independent inhibitory activity, either against trypsin or chymotrypsin (32). In contrast, SFTI-1 consists of only 14 amino acid residues and has only one inhibitory loop. Its small size leads to the suggestion that SFTI-1 is possibly derived from a "normal size" BB inhibitor as a result of peptide bond cleavage followed by a transpeptidation reaction or a peptide bond double cleavage followed by a ligation reaction. A likely location for the joined ends of cyclic SFTI-1 is the D14G1 position within the non-binding loop (Fig. 8). The detection of an iso-aspartic acid residue in position 14 of one of the structural species of SFTI-1[6,5] is an indication of the high reactivity of this functional residue and supports the possibility of participation of this residue in a transpeptidation reaction. However, up to now, no putative precursor protein of the BB inhibitor family is known with D and G or alternatively N and G in the relevant positions.
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From the existence of the iso-Asp residue a third alternative suggestion for in vivo cyclization of SFTI-1 is conceivable, starting from the putative precursor SFTI-1[1,14] (Fig. 8). In general, peptide bonds are subject to both acid and base hydrolysis. Most peptide bonds are stable except those in Asp-Xaa sequences, with cleavage being particularly rapid at DG and DP bonds due to a reduced energy barrier (42). Because peptide bond hydrolysis/synthesis is an equilibrium reaction, it can theoretically occur in both directions. For linear peptides the reversal reaction is highly unlikely due to separation of the reaction products. However, formation of the D-G bond in the case of a linear SFTI-1 precursor open between G1 and D14 leading to cyclic SFTI-1 is not as difficult, because both termini are in close proximity due to the disulfide bond and the continuous sequence of the putative precursor. Because the side-chain carbonyl group of D14 is involved in a hydrogen bond with the amide proton of R2, it is available for a nucleophilic attack of the amide nitrogen of G1 for succinimide formation. Hydrolysis on either side of the succinimide nitrogen atom results in an D or iso-D residue within the peptide chain. A feasible mechanism for cyclization of SFTI-1 from SFTI-1[1,14] is the formation of a cyclic anhydride within D14, which would lead after nucleophilic attack of G1 to a peptide bond between D14 and G1. However, this reaction does not seem to occur spontaneously as no cyclic product was detected in our investigation of this putative precursor of SFTI-1 (11) indicating that a catalytic enzyme might be necessary. Alternatively, a COOH-terminal N instead of D residue within a putative precursor peptide could form an intra-residual succinimide that might more readily be attacked by the NH2-terminal G1 than the cyclic anhydride of aspartate. Subsequent deamidation of the asparagine residue would lead to the formation of cyclic SFTI-1 with its DG peptide bond. Interestingly, the cyclic inhibitor MCoTI-II from Momordica cochinchinensis exhibits also an DG peptide bond that gives rise to two isomers of MCoTI-II (9).
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CONCLUSIONS |
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Tables S1 and S2, and additional Refs. 15.
** An ARC Senior Fellow. To whom correspondence should be addressed. Tel.: 61-7-3346-2019; Fax: 61-7-3346-2101; E-mail: d.craik{at}imb.uq.edu.au.
1 The abbreviations used are: SFTI-1, sunflower trypsin inhibitor-1; BB, Bowman-Birk; DQF-COSY, double quantum filtered two-dimensional correlation spectroscopy; E.COSY, exclusive COSY; LC-MS, liquid chromatography mass spectrometry; NOE, nuclear Overhauser effect, also used for NOESY cross-peak; NOESY, two-dimensional NOE spectroscopy; TOCSY, two-dimensional total correlation spectroscopy; WATERGATE, water suppression by gradient-tailored excitation; r.m.s.d., root mean square deviation.
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REFERENCES |
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